DNA Binding Domains and Nuclear Localization Signal of LEDGF: Contribution of two Helix-Turn-Helix...

doi:10.1016/j.jmb.2005.10.054 J. Mol. Biol. (2006) 355, 379–394

DNA Binding Domains and Nuclear Localization Signalof LEDGF: Contribution of two Helix-Turn-Helix (HTH)-like Domains and a Stretch of 58 Amino Acids of theN-terminal to the Trans-activation Potential of LEDGF

Dhirendra P. Singh1*, E. Kubo2, Y. Takamura1, T. Shinohara1, A. Kumar3

Leo T. Chylack Jr4 and N. Fatma1

1Department of Ophthalmologyand Visual Sciences, Universityof Nebraska Medical CenterOmaha, NE 68198, USA

2Department of OphthalmologyUniversity of Fukui, Fukui9101193, Japan

3Department of MicrobiologySchool of Medicine, PonceSchool of medicine, Ponce USA

4Center for OphthalmicResearch, Brigham andWomen’s Hospital, HarvardMedical School, Boston, MA02115, USA

0022-2836/$ - see front matter q 2005 E

Abbreviations used: LEDGF, lensgrowth factor; NLS, nuclear localizahelix-turn-helix; GFP, green fluorescglutathione-S-transferase; PWWP, ptryptophan-proline; HSE, heat shocstress response element; Hsp27, heaamino acid residue(s); EMSA, electrshift assay; CAT-ELISA, chloramphase-enzyme-linked immunosorbent

E-mail address of the [email protected]

Lens epithelium derived growth factor (LEDGF), a nuclear protein, plays arole in regulating the transcription of stress-associated genes such as heatshock proteins by binding to consensus core DNA sequences nAGGn ornGAAn or their repeats, and in doing so helps to provide cyto-protection.However, additional information is required to identify the specificstructural features of LEDGF involved in gene transcription. Here wehave investigated the functional domains activating and repressing DNA-binding modules, by using a DNA binding assay and trans-activationexperiments performed by analyzing proteins prepared from deletionconstructs. The results disclosed the DNA-binding domain of N-terminalLEDGF mapped between amino acid residues 5 and 62, a 58 amino acidresidue stretch PWWP domain which binds to stress response elements(STRE; A/TGGGGA/T). C-terminal LEDGF contains activation domains,an extensive loop-region (aa 418–530) with two helix-turn-helix (HTH)-likedomains, and binds to a heat shock element (HSE; nGAAn). A trans-activation assay using Hsp27 promoter revealed that both HTH domainscontribute in a cooperative manner to the trans-activation potentialof LEDGF. Interestingly, removal of N-terminal LEDGF (aa 1–187)significantly enhances the gene activation potential of C-terminal LEDGF(aa 199–530); thus the N-terminal domain (aa 5–62), exhibits auto-transcriptional repression activity. It appears that this domain is involvedin stabilizing the LEDGF–DNA binding complex. Collectively, our resultsdemonstrate that LEDGF contains three DNA-binding domains, whichregulate gene expression depending on cellular microenvironment andthus modify the physiology of cells to maintain cellular homeostasis.

q 2005 Elsevier Ltd. All rights reserved.

Keywords: lens epithelium-derived growth factor; DNA-binding domain;transcription–regulation; stress and heat shock elements; PWWP domain
*Corresponding author
lsevier Ltd. All rights reserve

epithelium-derivedtion signal; HTH,ent protein; GST,roline-tryptophan-k element; STRE,t shock protein 27; aa,ophoretic mobilityenicol acetyltransfer-assay.ing author:

Introduction

The regulation of eukaryotic transcriptionis accomplished by multiple sequence-specificprotein–DNA interactions. The DNA-binding pro-tein domains, which recognize their cognate DNAelement(s) in the promoters of genes, act on othertranscriptional factors and eventually turn on andoff transcriptional initiation or act as enhancers orrepressors of transcription.1,2Eukaryotic transcrip-tional regulators obviously play a role in the

d.

380 DNA-binding Domains of LEDGF

nucleus, where they activate or repress geneexpression. Nuclear localization can be an essentialstep in transcriptional regulation. Thus, mosttranscription factors bear nuclear localizationsignal(s) (NLS) or utilize other proteins with NLSas a vehicle to travel to nuclei. Various mechanisms,including reversible phosphorylation of sequencesadjacent to the NLS, unmasking of the NLS, orrelease from an inhibitory protein, can result ininducible nuclear localization.3 However, lensepithelium derived growth factor (LEDGF) ispredominant in the nucleus and found in mostcells.4–6 A stress-inducible transcriptional survivalfactor,7 LEDGF belongs to the hepatoma-derivedgrowth factor family of proteins that consists of awell-conserved N-terminal amino acid sequencecalled the HATH (homologous to amino terminusof HDGF) region8,9 with a PWWP domain (proline-tryptophan-tryptophan-proline). Its gene yieldstwo proteins: LEDGF/p75 and p52, by alternativesplicing.10 P52 is a general co-activator of transcrip-tion11 and also a co-activator of the splicing ofpolymerase II-restricted mRNAs. In vitro as well asin vivo, cells overexpressing LEDGF, such as lensepithelial cells (LECs), rod-photoreceptor cells,corneal epithelium, Cos7 cells, skin fibroblasts,and keratinocytes exhibit increased resistance toenvironmental stress and improved survival.12–14

Interestingly, these cells display elevated expres-sion levels of small Hsp27 and a-B-crystallin,suggesting a mechanism for LEDGF-mediatedprotection.4,15 LEDGF up-regulates stress-associated genes by binding to the stress response(STRE; A/TGGGGA/T) and heat shock elements(HSE; nGAAn) present in these genes.5,10,16,17

Recently, the involvement of LEDGF has beenreported in human immunodeficiency virus type-1(HIV-1) integration,18 autoimmune disorders,19–22

and cancer by the formation of a chimaeric proteinwith NUP 98.23,24 In addition, evidence has revealedthat LEDGF plays a role in lens epithelial to fibercell terminal differentiation.6 More recently, thepresence of this molecule in discrete regions andcell types within the fetal and adult brain has beenreported, which suggests that it may be involved inneuro-epithelium stem cell differentiation andneurogenesis.25 Furthermore, the involvement ofLEDGF/p75 has been reported in the prevention ofproteosomal degradation of HIV-1 integrase.26

These studies have underscored the wide range ofroles played by LEDGF, ranging from cellularprotection to association with cellular abnormal-ities. The mechanisms by which LEDGF is involvedin various cellular events and the specific ways inwhich it plays its diversified roles are subjects ofactive investigation. We have predicted that thefunctions of LEDGF are associated with thepresence of different functional domains in itsproteins that interact with DNA and/or proteins,and thus regulate gene transcription. Dysfunctionof the LEDGF protein would be expected to leadto disturbed cellular physiology.27,28 Moreover,it has been demonstrated that the activation of

ERK signaling is involved in regulating LEDGF.29

A high expression level of LEDGF/p75 inprostate tumors and benign prostate hyperplasia hasbeen reported, and it is suspected that the cleavagefragments generated by caspases might triggerauto-antibodies under conditions of inflammation.30

Moreover, LEDGF is a nuclear protein and bindsto DNA.5,10,16,17,27,31 This protein bears a singleclassical NLS (148-GRKRKAEKQ-156) in theN-terminal region, and four central basic residuesin the NLS are critical for its activity.32–35 In earlierstudies, we have shown that LEDGF binds to anheat shock element (HSE) and stress responseelements (STRE) in the promoters of stress-associated genes such as Hsp27 and aB-crystallin,and activates their transcription.4,5,10,16,17,27 Theconsensus core nucleotide sequences to whichLEDGF binds are GAA and AGG or their repeats.LEDGF may bind to a single core sequence or tomultiple core sequences, such as nGAAnnTTCn orAGGGGA. We have reported that LEDGF alsobinds to AGGGA sequences.10 Recently similarobservations have shown its affinity to DNAcontaining AGGGA sequences.36 Here, weattempted to define LEDGF binding sequencesdivergent from its core binding sequences,A/TAGG and/or nGAAn. Moreover, LEDGF, ahighly charged lysine and arginine-rich protein,binds to heparin and DNA. The N-terminal LEDGFcontains a PWWP domain that spans 58 amino acidresidues (aa 5–62), with a conserved PHWP motif.Notably, the Dnmt3b PWWP domain has beenshown to bind DNA in vitro,37 but most of theseresidues are not conserved.37–39 Despite the knownregulatory function of LEDGF, investigation intothe molecular structure–function relationship ofLEDGF’s domains has been limited.

Bioinformatic analysis (Network ProteinSequence Analysis, IBCP (NPS@, IBCP)) revealedthe presence of two HTH domains and an extensiveloop in the C-terminal region of LEDGF. Theseregions are significantly associated with the tran-scriptional regulatory function of LEDGF.40,41 Web-based analysis of the LEDGF protein using theSimple Modular Architecture Research Tool(SMART),42,43 which showed the presence of AThook-like (HMG) and coiled-coil domains, furtherargues for the involvement of this protein in generegulation. Based on our previous work,5,10,16,17,27

we believe that LEDGF’s trancriptional activitymight be associated with interaction(s) of differentfunctional domains of LEDGF with different DNAelements. Therefore, to examine the molecular basisof LEDGF function, we analyzed functional DNA-binding domains that may recognize two indepen-dent DNA target sequences, as would be necessaryfor stress-responsive gene regulation. Further ana-lysis of the LEDGF protein (Network ProteinSequence analysis, IBCP (NPS @, IBCP andSMART)) indicated that this molecule has threepotential DNA-binding domains: a PWWPstretch of 58 amino acid residues (aa 5–62) andtwo helix-turn-helix (HTH 1–421-YNKFKNMFLV-

DNA-binding Domains of LEDGF 381

GEGDSVITQVLN-442, and HTH 2–471-QTGSKTLNGGSDAQDGNQPQHN-492) domains in theN and C-terminals of LEDGF, respectively.

Here we describe the transcriptional activationpotential of functional domains of LEDGF and itsNLS revealed by the introduction of a variety ofdeletion mutations. We identify three novel activationdomains of LEDGF, one auto-inhibitory domain inthe N-terminal and two activating HTH-like domainsin the C-terminal. We also identify the contributionsto the trans-activation potential of each domain.Defining the cooperative or non-cooperative func-tions of these domains in the regulation of the genepromoter may make it possible to explore LEDGF’sroles in cellular survival as well as in carcinogenesisand AIDS. We also describe the binding affinity ofLEDGF to various consensus core sequences byaltering the positions of the bases, which may permitfurther characterization of LEDGF’s functionaldomain(s) and should provide insights into themolecular mechanism(s) through which LEDGFexerts its diverse effects.

Results

Bioinformatic analysis of DNA binding domainsof LEDGF protein

To investigate the structural organizationof LEDGF, we utilized a Web-based computerprogram, Simple Modular Architecture Tool(SMART) to predict the putative domains of

CI

CII

C

PWWP

199

(b)

NH2

1NLS

(a)

5 62

178 197

Loop I

177 250

Coiled coil

306 334

Lys rich rAT hook like

NI

NII

NIII

NIV

PWWP NLS

146 158

1 187

PWWP1 135

PWWP1 101

PWWP1 68

PWWP1 35NV

(c)

NVI PWWP NLS1 250

CVII170

Figure 1. (a) Schematic representation of computer-preinteraction sites (aa 5–62); NLS, nuclear localization signal (aaCoiled coil (aa 306–334); IBD, integrase binding domain (aa421–442); HTH2 (aa 471–492); Loop 2 (aa 440–530). (b) Plasmidof LEDGF. NI, aa 1–187; NII, 1–135; NIII, 1–101; NIV, 1–68; NVLEDGF. CI, aa 199–530; CII, 302–530; CIII, 322–530; CIV, 34constructs were generated using PCR with LEDGF-specific seGFP vectors for intracellular localization as well as CAT-ELprotein was expressed, purified, and used for DNA binding

LEDGF/p75. Results of the search displayed thepresence of a PWWP domain, a stretch of 58 aminoacid residues (aa) at the N-terminal of LEDGFspanning from aa 5 to aa 62. C-terminal LEDGFconsists of two HTH domains (HTH1, aa 421–442;HTH2, aa 471–492) (Figure 1(a)). In addition,computer analysis further revealed the presence ofan AT-hook-like domain (aa 178–197), and a coiled-coil domain (aa 306–334). An analysis using theNORD program disclosed the presence of loopystructures at two sites, loop1 (aa 177–250) and loop2(aa 440–530) (Figure 1(a)), and this dual loopedstructure is implicated in DNA binding. In addition,five low complexity regions (data not shown) anda lysine-rich region profile (aa 216–343) werepredicted in the LEDGF protein.

Identification of the N-terminal recognitionsequence

Previously, we reported that full LEDGF proteinconstitutively and functionally binds to HSE andSTRE present in the stress response genes.10

Here, we defined the distinct domains of LEDGF.Based on computer prediction of DNA-bindingdomains of LEDGF (as mentioned above), we madevarious fusion constructs linked to GST or GFPvectors, as described in Materials and Methods andFigure 1(b) and (c). The fusion proteins wereexpressed and purified.44 To determine LEDGF’sN-terminal DNA binding element, we used NI (aa1–187) in affinity chromatography as described10

(Materials and Methods). Briefly, the purified

III

CIV

CV

CVI

530 amino acidsHTH 1 HTH 2 COOH

440 530

421 - 442

471 492

Loop II

egion

HTH 1 HTH 2530

HTH 1 HTH 2530 302

HTH 1 HTH 2530 322

HTH 1 HTH 2530 348

HTH 1 HTH 2530 418

HTH 2530 443

HTH 1 HTH 2530

347 429IBD

dicted functional domains of LEDGF. PWWP, protein146–158); Loop I (aa 177–250); AT-hook-like (aa 178–197);347–429); HTH, helix-turn-helix DNA binding motif (aaconstruction of truncated forms of the N-terminal region

, 1–35; NVI, 1–250. (c) Truncated C-terminal constructs of8–530; CV, 418–530; CVI, 443–530; CVII, 170–530. Thesense and antisense primers and cloned into pEGFP and/orISA. These were also cloned into a pGEX2T vector, andassay.

Table 1. Alignment of sequences of N-terminal LEDGF binding oligos, isolated through CNBr Sepharose-4B columnusing a Weight Matrix/Nucleotide Distribution Matrix Programme (Genomatic)

Aligned sequences of N-terminal LEDGF binding oligos isolated through LEDGF-CNBr Sepharose-4B column1. - -TGATAGGGGATG- - - 21. - - -TTGTGGGGTCCT-2. TATTTTAGGGGA- - - - - 22. - -GTGCTGGATAGG- - -3. - -ACTGAGGGGAGG- - - 23. - -GTGCTGGATAGG- - -4. TCACTGAGGGGA- - - - - 24. -TACGCTAGGCCG- - - -5. TTTAGGAGGGGC- - - - - 25. -GACGTCGGGCCC- - - -6. -GGATGAGGGGTT- - - - 26. - -CGGTTAGGTAAG- - -7. - -TCCTGGGGTCGG- - - 27. - -CGGTTAGGTAAG- - -8. - -TTGGGGAGTAGG- - - 28. - - - -GTTGGGTGCGTG-9. - - - -TAATGGGAGGAA- 29. - - - - -GTGGTAATGGGG10. TTATGTTGGGGA- - - - - 30. - -- -TGAGGTTATGTG-11. GTGCGGTGGGGT- - - - - 31. - - - -TGAGGTTATGTG-12. -TGTGGTGGGGTG- - - - 32. - - - - -TGAGGTTATGTG-13. -TGTGGTGGGGTG- - - 33. - -ACCTTGGAACCG- - -14. -TGTGGTGGGGTG- - - - 34. - -ACCTTGGAAACC- - -15. -TGTGGTGGGGTG- - - - 35. - - - - -TTGGCCGTCATG16. -GGCGGTGGGGAA- - - - 36. - - -CTAGGAGGCAGA- -17. -GGCGGTGGGGAA- - - - 37. - - -CTAGGAGGCAGA- -18. -GGCGGTGGGGAG- - - - 38. - - - -GTGGAGGCCGAG-19. -GGCGGTGGGGTA- - - - 39. - - - -GTGGAGGCCGAG-20. - - -TTGTGGGGACCT- - 40. - - - -GTGGAGGCCGAG-Sequence conservation among 40 clones selected for LEDGF binding

Position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

A 0 1 7 2 1 3 12 3 6 4 10 20 7 0 7 1 1C 0 1 2 14 3 3 1 0 0 1 3 7 9 4 0 0 0G 1 5 12 3 22 20 6 36 34 31 24 5 12 20 1 6 2T 4 8 5 11 12 14 21 1 0 4 3 8 7 0 6 1 7SUM 5 15 26 30 38 40 40 40 40 40 40 40 35 24 14 8 10

ConsensusSequences:

T T G/A C/T G/T G/T T G G G G A N G W G T

Affinity chromatography was carried out using the method of Oliphants et al.64 N-terminal protein (NI aa, 1–187) was covalently boundto a CNBr-activated Sepharose 4B column. A double-stranded radiolabeled probe having a randomized sequence of 12 nucleotides wasadded to the column. After three rounds of selection, bound oligos were eluted, amplified, and cloned in Bluescript, and DNAsequences were determined. Alignment of 40 sequences resulted in the consensus sequence TGGGGA, as indicated.


protein NI was covalently bound to a CNBr-activated Sepharose-4B column and exposed torandom 12-mer nucleotides. Oligos bound to NIwere eluted and sequenced following cloning. Theresults revealed that NI bound to a consensus coresequence of A/TGGGGA/T (STRE) (Table 1). Weverified this result using electrophoretic mobilityshift assay (EMSA) and found that N-terminalLEDGF indeed bound to the STRE sequence(Probe; P1) (Table 1 and Figure 2(a), lane 1) butnot to nGAAn or nAGGn (Probes; P2 and P3)(Figure 2(a); lanes 4 and 7). Notably, full LEDGF (aa1–530) bound to nAGGn or nGAAn or theirrepeats,10 while in contrast, the N-terminal did notbind to nAGGn on EMSA, suggesting that theN-terminal requires repeats of nAGGn. Possibly aspecific conformation of the LEDGF protein isrequired for binding to nAGGn. N-terminalLEDGF loses its conformation and requiresnAGGn repeats to bind.

A stretch of 58 amino acid residues, the PWWPdomain, is essential for binding to A/TGGGGA/Tand trans-activation of stress-associated genes

Next, we wished to determine the minimum aminoacid residues required for STRE (A/TGGGGA/T)binding. We engineered various truncated constructs

(NII, NIII, NIV, and NV) of N-terminal LEDGF(Figure 1(b)). Bacterially expressed recombinantproteins (Figure 2(b) and (d)) were used to performEMSA and incubated with a 32P labeled probe(double-stranded DNA) containing the STREsequence. All peptides did bind to the STREsequence (Figure 2(b), lanes 3 and 5; Figure 2(d),lanes 1 and 3). However, NV (aa 1–35) bound onlyweakly to STRE (Figure 2(d), lane 1). From thisexperiment, a PWWP domain spanning from 5 to 62amino acid residues was defined, one which boundspecifically to the STRE element (P1 probe)(Figure 2(d), lane 3) but not to HSE (P3 probe)(Figure 2(d), lane 7).

To study whether the binding of N-terminalLEDGF peptides is functional, we conductedchloramphenicol acetyltransferase-enzyme-linkedimmunosorbent assay. (CAT-ELISA) using theHsp27-gene promoter or its mutant at STRE linkedto a CAT reporter vector, and the activity wascompared with that of full-length LEDGF (Figure 4).Transfection experiments using NI constructdemonstrated significant CAT activity of Hsp27.However, other constructs, NII, NIII, and NIV,showed quite similar effects on trans-activation.NV (aa 1–35) was not able to significantly trans-activate gene transcription (Figure 4). These resultsindicate that deletion of amino acid residues

Figure 2. (a) N-terminal LEDGF binds to STRE (A/TGGGGA/T; P1) but not to (nAGGn; P2 or nAGAAn; P3)consensus sequences. 32P-labeled oligos were incubated with N-terminal (NI) protein (aa 1–187) at 4 8C for 30 min andthen subjected to electrophoresis. Oligos containing the stress response consensus sequence (A/TGGGGA/T) formed aspecific complex with NI (lane 1) and supershifted when LEDGF-specific antibody was added (Ss1). However,



beyond position 68 significantly abolished thetrans-activation activity of the PWWP domain.Thus, a stretch of 58 amino acid residues (aa 5–62)is essential for binding and trans-activation.Furthermore, STRE mutant Hsp27 did not showtranscriptional activity in the presence of any ofthe N-terminal constructs, demonstrating that theN-terminal binding domain trans-activates theHsp27 gene promoter by binding to STRE. Incontrast, C-terminal LEDGF (CI) could activate theHsp27 promoter mutated at the STRE site (Figure 4,dotted bar).

Both C-terminal helix-turn-helix domains areessential for DNA-binding and trans-activation

The findings described above show theN-terminal LEDGF binding to STRE. We hypo-thesized that the C-terminal may bind to HSE(nGAAn). To test this, we carried out EMSA usingthe C-terminal and its deletion derivatives. We usedthe proteins derived from different constructs toperform EMSA using radiolabeled oligomerscontaining HSE elements. Results demonstratedthat CI (aa 199–530) bound to nGAAn and nAGGn(Figure 5(a), lanes 1 and 4) but not to A/TGGGGA/T(Figure 5(a), lane 7). We verified these results usingan anti-LEDGF antibody that further retards theCm1 complex to an Ss1 complex (Figure 5(a), lanes 2and 5). The addition of a cold competitor eliminatedthe Cm1 bands (Figure 5(a), lanes 3 and 6).

Next, to determine whether the HTH domains atpositions 421–442 and 471–492 are responsible forthe binding of the C-terminal LEDGF to nGAAn, weutilized the constructs CIII, CV, and CVI in EMSA(Figure 5(b)). CIII and CV contain two HTHdomains, but CVI contains only one (Figure 1(c)).CIII and CV bound to nGAAn (Figure 5(b), lanes 1and 3), but CVI, having only one HTH, did not bindto this probe (Figure 5(b), lane 5).

We then conducted trans-activation experimentsto assess the activation potential of each construct ofC-terminal LEDGF. Using a transfection assay withHsp27 promoter-CAT or its HSE mutant constructalong with LEDGF’s C-terminal constructs, wemonitored the CAT activity of the Hsp27 promoter

N-terminal LEDGF did not bind to the oligos having HSE, nGLEDGF binds to STRE (P1) but not to HSE (P3). A gel-shift assaN-terminal LEDGF (NI, NII and NIII) and a 32P-labeled probebind to probe P1 (lanes 1, 3, and 5). At the same time, NI did nNH2 terminus of LEDGF binds specifically to the A/TGGGGAmigrate similarly, likely as a result of a significant loss of nterminus. (c) Western analysis showed that the protein studexpressed proteins contain a GST flag (w29 kDa). (d) A stretbinds to STRE (P1) but not to HSE (P3). A gel-shift assay wLEDGF (NIV and NV) and a 32P-labeled probe having HSE ahaving STRE (lanes 1 and 3), and the specific Cm1 band cou(lanes 2 and 4), but did not bind to P3 having HSE (lanes 5residues in the NH2 terminus of LEDGF binds specificallyrecombinant proteins derived from these constructs reveals thcontain a GST flag (w29 kDa).

with CAT-ELISA. We found that any construct fromCI to CV containing both the HTH domains couldtrans-activate the promoter activity of Hsp27. Incontrast, CVI, having only one HTH domain, couldnot trans-activate the Hsp27 promoter (Figure 6).A higher level of promoter activity was observed inCI (aa 199–530) than in full LEDGF and the otherconstructs. Furthermore, mutation at HSE couldabolish the trans-activation of Hsp27 gene promoterby the C-terminal LEDGF, suggesting that theC-terminal LEDGF activates the stress-associatedgenes by binding to HSE present in these genes(Figure 6), while the N-terminal (NI) could activatethe promoter (Figure 6, stippled bar).

AT-hook-like sequence does not affect DNAbinding activity of LEDGF

We performed EMSA using N and C-terminalLEDGF proteins containing the AT-hook-likedomain. Results demonstrated that the N-terminalLEDGF-NVI (aa 1–250) bound to the probe contain-ing A/TGGGGT/A (Figure 3(a), lane 3) but not tothe probes with HSE, nGAAn, or the AT-richsequence (Figure 3(a), lanes 1 and 5). C-terminalLEDGF-CVII (aa 170–530) bound to the probe withnGAAn (lane 7) but did not bind to A/TGGGGT/Aor the AT-rich probe (lanes 9 and 11). The resultsindicate that the AT-hook motif did not participatein or interfere with the binding activity of LEDGFprotein.

Contribution of N and C-terminal LEDGF in thetrans-activation potential of LEDGF

The results described above showed that theC-terminal had a higher ability to trans-activate thepromoter activity compared to full or N-terminalLEDGF, and this activity was about fourfold higherthan that of the N-terminal (Figures 4–6). TheN-terminal of LEDGF, the PWWP domain, appearsto control the regulation of LEDGF by inhibitingits trans-activation and functions as the auto-repressive/inhibitory domain of LEDGF. LEDGFregulation of stress-associated genes may beassociated with the physiological state of cells. In

AAn, or nAGGn (lanes 4 and 7). (b) The NH2 terminus ofy was performed using various truncated proteins of GST-having HSE and STRE binding elements. NI, NII, and NIIIot bind to P2 (HSE) (lane 7). This further confirms that the/T sequence. On the native gel, NI, NII, and NIII appear toegative charge accompanying the deletion of the COOHied exhibited the appropriate molecular mass. Note: allch of 58 amino acid residues in NH2 terminus of LEDGFas performed using truncated protein of GST-N-terminalnd STRE binding elements. NIV and NV bind to probe P1ld be eliminated by addition of an unlabeled competitorand 7). This result shows that a stretch of 68 amino acidto the A/TGGGGA/T sequence. (e) Western analysis ofe appropriate molecular mass. Note: all expressed proteins

Figure 3. The AT-hook-like domain of LEDGF does not affect DNA binding activity of LEDGF. (a) A gel-shift assay wasperformed using NVI (aa 1–250) and CVII (aa 170–530) containing AT-hook-like domain. NVI formed specific complex(Cm1) with the probe containing A/TGGGGA/T (P1) (lane 3) but did not bind to the probes containing nGAAn (P3) orAT-rich sequences (P4) (lanes 1 and 5). CVII bound to nGAAn (lane 7) but not to the probe having A/TGGGGT/A orAT-rich sequences (lanes 9 and 11). (b) Western analysis demonstrating the molecular mass of the proteins used in gel-shift assay.


other words, cells require a certain amount of geneproducts for their survival at the physiologicallevel, and we suggest that the N-terminal domainplays an important role in genetic expression, viainteraction with other proteins and/or by stabil-izing the DNA–protein complex.

–214

471 492

Loop II

NIPWWP NLS 187

1

1

PWWP1 135

PWWP1 101

PWWP1 68

PWWP1 35

NII

NIII

NIV

NV

VectorEmpty

(a) (b)

PWWP NLS 1871NI

0

530

1

HTH 1 HTH 2CI 199530

PWWP NLS HTH 1

421 -442

HTH 2

Figure 4. Transcriptional activation by various truncaterepresentation of different partial constructs of N-terminalInvitrogen) vector for eukaryotic expression (left panel). (b) Tof LEDGF (NI, NII, NIII, NIV and NV). Cells were co-transfectwith the Hsp27 gene promoter linked to CAT (pCAT basic vextracted. CAT-ELISA was performed and CAT activity wasusing a pSEAP basic vector (Fatma et al.)17 and then comparepresent means GSD from three independent experiments.

Both, N and C-terminal domains of LEDGF areessential to engender the resistance againstoxidative stress

The potential of LEDGF and its deletion mutantsNI and CI to protect cells against paraquat-induced

+21HSE STRE–185 –178 –114 –106CAT

Relative Transactivation Activity (%)40 80 120

STRE–114 -106

X

d proteins of NH2 terminus of LEDGF. (a) SchematicLEDGF generated in a pEGFP and/or GFP (Clontech,

he trans-activation activities of five N-terminal constructsed with either of the indicated constructs of LEDGF alongector). After 72 h, cells were harvested and protein wasrecorded. The transfection efficiencies were normalized

red with that contributed by the empty vector. The data

Figure 5. (a) The C terminus LEDGF binds to HSE. EMSA was performed with the recombinant C-terminal LEDGF(CI; aa 199–530) and 32P-labeled probes containing HSE; nGAAn (P3), nAGGn (P2), and STRE; A/TGGGGA/T (P1). CIbound to P3 and P2 (lanes 1 and 4) but not to P1 (lane 7). Addition of LEDGF antibody supershifted the Cm1 bands to Ss1bands (lanes 2 and 5), and addition of cold competitor eliminated the Cm1 bands (lanes 3 and 6), showing the specificityof binding. (b) The COOH terminus of LEDGF (CIII and CV) bearing two HTH binds to HSE. EMSA was performed withtruncated recombinant C-terminal LEDGF, CIII (aa 322–530), CV (aa 418–530) and CVI (aa 443–530), and a 32P-labeledprobe containing HSE (P3). CIII and CV bound to the probe and made the Cm1 band (lanes 1 and 3), but CVI did not bindto the same probe (lane 5). The addition of unlabeled self competitor at a 1000-fold molar excess eliminated the Cm1band (lanes 2 and 4). (c) Western analysis demonstrates that the expressed protein exhibited the appropriate molecularmass. Note: all expressed proteins contain a GST flag (w29 kDa).


oxidative stress was monitored. Figure 7 (gray bar)reveals that cells transfected with vector aloneunderwent oxidative stress-induced death. Weshowed earlier that the cellular death caused byoxidative stress in LECs is apoptotic.27,28 Cells

transfected with NI- or CI-LEDGF constructsshowed less resistance against oxidative stress(Figure 7, stippled bar and stripped bar, respect-ively), while LEDGF-transfected cells survived wellin similar conditions (black bar). Thus, our results

–214 +21HSE STRE–185 –178 –114 –106CAT

Relative Transactivation Activity (%)

CI

CII

CIII

CIV

CV

CVI

HTH 1 HTH 2

HTH 1 HTH 2302

HTH 1 HTH 2322

HTH 1 HTH 2348

HTH 1 HTH 2418

HTH 2443

199

Empty Vector

PWWP NLS HTH 1 HTH 2

421 - 442

471 492

Loop II1

530

(a) (b)0 40 80 120 160

HSE–185 –178X

HTH 1 HTH 2CI 199

PWWP NLS1871NI

Figure 6. Transcriptional activation by various truncated proteins of the C terminus LEDGF. (a) Schematicrepresentation of different partial constructs of C-terminal LEDGF generated in pEGFP and/or GFP vector foreukaryotic expression (left panel). (b) The trans-activation activities of the truncated constructs of LEDGF (CI, CII, CIII,CIV, CV, and CVI). Cells were co-transfected with either of the indicated constructs along with the Hsp27 gene promoterlinked to the CAT vector. After 72 h, cells were harvested and protein was extracted. The transfection efficiencies werenormalized using a pSEAP basic vector and values were compared with those contributed by the empty vector. The datarepresent the meanGSD from three independent experiments.


demonstrate that both N- and C-LEDGF contributeto the protective potential of LEDGF during stress.

0

20

40

60

80

100

120

140

160

F-LED NI-LED CI-LED Vec

Num

ber

of n

ucle

i

Figure 7. Histogram showing the effect of LEDGF andits N and C-terminal domains on the LEC survival facingoxidative stress. Cells were stably transfected with EGFP-full-LEDGF (F-LED), with EGFP-NI-LEDGF (NI-LED),with EGFP-CI-LEDGF (CI-LED), or with EGFP-vector(Vec) to monitor their protective potential effect againstoxidative stress. These cells were cultured in DMEMcomplete medium, and 24 h later, subjected to paraquat-induced oxidative stress (2 mM) (Materials and

Nuclear localization signal of LEDGF

To identify the sub-cellular localization pattern ofthe LEDGF constructs prepared (Figure 1), we usedthree cell lines: Cos7 cells, fibroblasts, and LECs.Figure 8(a) represents Cos7 cells transfected withthese constructs. Results were identical to thefindings reported earlier.34,35 Full-length GFP-LEDGF (aa 1–530) and NI (aa 1–187) localizedexclusively in the nucleus (Figure 8(a), a and b). Incontrast, neither the CI construct nor NII (aa 1–135)were present in the nucleus (Figure 8(a), CI andNII), suggesting that NLS is present between aminoacid residues 136–187. As shown in Figure 8(b), wechanged R46 to I46, and R49 to I49 (Mut1), but thispoint mutation did not abolish NLS, and the mutantconstruct localized to the nucleus (Mut1). Next, inaddition to this mutation, we changed R51 to I51(Mut2). Interestingly, this mutation did abolishthe nuclear localization of LEDGF (Mut2). To assessthe importance of the specific amino acid residues,we made point mutations at K55 to I55 (Mut3;R49 to I49, R51 to I51, and K55 to I55) that furtherinhibited the localization of LEDGF to nucleus.These results suggest that R49, R51, and K55 are themajor amino acid residues responsible for thenuclear localization of LEDGF (Figure 8(b) and (c)).

Methods). Then, 72 h later, DAPI staining was conductedand cells were counted under fluorescence microscope inrandomly selected fields. The histogram represents theaverage number of cells (five fields). Black bar, LEDGF;stippled bar, NI-LEDGF; striped bar, C1-LEDGF; gray bar,vector. Results demonstrate that both domains areessential for cellular survival during oxidative stress.

LEDGF binds to sequences that diverge from theconsensus binding sites

We were interested in the affinity and associationof LEDGF binding to sequences which diverge

from its core binding sequences. LEDGF binds toDNA constitutively and activates the gene.We obtained further evidence on the sequences-GAA- and -A/TGGGGA/T-, which have beenidentified as the unit of interaction with LEDGF.We synthesized oligos commercially containingvarious patterns of nAGGn and nGAAn, such as

Figure 8. (a) Flourescent images of Cos7 cells expressing full-length LEDGF and its deletion mutants. Cos7 cells weretransfected with each of the pEGFP and/or GFP-partial LEDGF constructs and the GFP vector (data not shown). Sub-cellular localization of expressed fusion proteins was visualized by confocal microscopy. a, LEDGF; b, NI; c, CI; d, NII. (b)Schematic representation of site-directed mutagenesis of the NLS site. Lysine (K) and/or argenine (R) were changed toisolucine (I) residues. (c) Fluorescent image of Cos7 cells transfected with a pEGFP-LEDGF-mutant in the NLS site. Sub-cellular localization of expressed fusion proteins (WT, Mut1, Mut2, and Mut3) was visualized by inverted fluorescentmicroscopy.


inverted repeat, palindromic, and tail-to-tail andhead-to-head sequences, as depicted in Figure 9.These probes were radiolabeled and used inEMSA with the full-length LEDGF protein. LEDGFhad a higher affinity to G2 (palindromic) andA/TGGGGA/T, STRE (head-to-head) and thelowest affinity to G5. Figure 9 shows that the othersequences, G3, G4, G6, G7, and G8, bound toLEDGF with almost equal affinity. From thesefindings, we concluded that LEDGF binds to DNAelements that diverge from its DNA binding sitesand may be associated with the domains that wehave identified in the present study. However, thefindings suggest that these divergent sequences arealso a target for LEDGF binding.

Discussion

Transcriptional factors are mostly sequence-specific DNA binding proteins that regulate geneexpression by either activating or repressing theinitiation of transcription. Mammalian trans-criptional factors are grouped into severalcategories based upon their structural domains forDNA binding and include the helix-turn-helix,

homeodomain, leucine zipper, and zinc fingerprotein families.45 Determination of how a parti-cular protein functions to coordinately regulate adiverse set of genes and establish a differentialphysiological state requires identification of itsfunctional domains. With this in mind, we soughtto identify the functional domains of LEDGF bypreparing mutant proteins and analyzing thesemutated proteins in DNA-binding and transienttrans-activation experiments. We found thatLEDGF consists of at least one N-terminal PWWP(aa 5–62) and two C-terminal HTH, aa 421–442(HTH I) and aa 471–492 (HTH II) transcriptionalactivation DNA binding domains, and a functionalNLS (aa 148–156) that is sufficient for translocationinto the nucleus. It became evident that theC-terminal domains are required for sequence-specific DNA-binding (nGAAn) (Figure 5(a)and (b)), because deletion of N-terminal LEDGF(aa 1–187) did not abolish the binding. Notably, asingle HTH domain alone does not bind(Figure 5(b)) and contribute to wild-type activity(Figures 5(b) and 6). Interestingly, N-terminalLEDGF binds to AGG repeats and/or the stressresponse element, A/TGGGGA/T (Figure 2(a), (b)and (d)). Further dissection of the N-terminal

Figure 9. A gel-shift analysis demonstrating LEDGF binding to sequences that diverge from the consensus bindingsite. Equivalent concentrations of radiolabeled probes containing the indicated sequences were incubated with equalamounts of GST-LEDGF protein, and binding was analyzed by gel electrophoresis. Specific protein–DNA complexes areindicated by the arrow (Cm1). The addition of unlabeled self-competitor at a 1000-fold molar excess eliminated the Cm1complex. Free probes appear at the bottom of the gel. The lower panel represents a histogram showing the relativebinding of different probes to LEDGF. Intensity of binding was estimated using pixels (web-based program, NCBI, NIH).


reveals a stretch of 58 amino acid residues, whichare essential for functional DNA-binding(Figure 2(d)). Thus, LEDGF consists of at leastthe three DNA-binding domains we have roughlydefined, which contribute to the trans-activationpotential of LEDGF. From the present study, itappears that at least two separate regions: thePWWP domain of the N-terminal and the two HTHdomains of the C-terminal, contribute in a coopera-tive manner to the trans-activation potential of theLEDGF protein. Regulation of gene transcription iscontrolled by transcriptional factors, which becomeactive depending upon the cellular state; therefore,transcriptional proteins acquire different assembliesof functional modules to regulate target genesdifferentially. Our results demonstrate that theLEDGF protein contains three distinct functionaldomains that are capable of regulating geneexpression and thus able to modify the physio-logical condition of cells depending upon thedemands of the cellular micro-environment. Wepropose that LEDGF controls gene transcription viaN-terminal repression modules in conjunction witha C-terminal activation domain in order to regulatestress-responsive genes. Thus, this study defines theregulatory DNA-binding domains of LEDGF thatmaintain the activity of stress-associated genes atnormal physiological levels.

Several lines of evidence have revealed thattranscription proteins bear repressive as well asactivation domains. It has been reported that dualDNA-binding domains, consisting of a paired set of

domains, each of which is comprised of two helix-turn-helix sub-domains, bind to distinct DNArecognition sequences and regulate differentially.46

The cGATA-1 protein47 contains two related fingerdomains, and the N-terminal finger plays a role inDNA binding by affecting the stability of the DNA–protein complex. We believe that the N-terminalLEDGF domain, PWWP, may similarly control genetranscription by stabilizing DNA and proteincomplex. Recently, PWWP has been reported toplay a role in gene regulation by binding to DNA.48

Dnmt3b PWWP has been shown to bind to DNAin vitro, probably through a surface area region thatis enriched with basic residues.37 Consistent withhaving a universal DNA binding function, PWWPdomains have been found, so far, only in nuclearproteins, some of which bind DNA,37 and weconsider LEDGF to be one of them. PWWP domainsare found in eukaryotic proteins, and are involvedin DNA methylation and DNA repair, including theregulation of transcription and transcription factorsinvolved in developmental processes. These obser-vations strengthen the suggested role of LEDGF intranscriptional processes and the regulation ofdevelopment. However, we found that LEDGF’sPWWP domain has a specific affinity to AGG coresequences (Table 1; Figure 2(a), (b) and (d)), andappears to be involved in modulating genetranscription.

Moreover, the underlying mechanism governingthe transcriptional regulation capacity and capa-bility of LEDGF, and the structural association that


might be responsible for the specific functionconferred by the domains of the N and C-terminalsof LEDGF, may lie in its PWWP and the HTH DNAbinding domains (Figures 4 and 6). LEDGF proteinhas been found to be highly susceptible to proteasesand caspases,41 suggesting most of the proteinexists as a flexible loop. The Engelman group wasnot able to detect interaction(s) of the PWWP andIBD domains, which further supports the claim thatthe domains of LEDGF are relatively independentin the full-length LEDGF.49 The regulatory functionof LEDGF may be associated with the flexibility ofthe LEDGF protein that is in accord with physio-logical state of cells. However, further work will berequired to address such concerns. Moreover,multiple or single AT-hook motifs have beendetected in a wide range of nuclear proteins indifferent species, including multi-domain chroma-tin-associated proteins involved in transcriptionactivation or suppression.50 Here, we found thatLEDGF does not bind to AT-rich sequences,suggesting that AT-hook-like domain of LEDGF isnot involved in its DNA binding activity (Figure 3)

In experiments to define sequences thatmediate the nuclear targeting of LEDGF, weidentified a single functional NLS in LEDGF,148

GRKRKAEKQ.156 Mutations of R-51-I and K-55-Iwere sufficient to block LEDGF nuclear localization.A similar finding was obtained by anothergroup.34,41 It has been reported that in 67% ofproteins that possess both DNA binding and NLSactivities, the NLS overlaps with the DNA bindingsequence.51 Subsequently Cokol et al.52 extendedthe data base search and expanded this ratio to90%. Our finding suggests that the NLS domaindoes not overlap with the DNA binding domainof LEDGF, since deletion mutants did efficientlybind to the A/TGGGGA/T element. Moreover,other laboratories have recently reported thepresence of LEDGF in the peri-nuclear area andhair follicles.22,25 These findings suggest thatLEDGF may have other functions. We earlierreported the nuclear disappearance of nuclearLEDGF during differentiation and in terminallydifferentiating cells.5,6

A mobility assay revealed that LEDGF binds tosequences divergent from nGAAn or A/TGGGGA/Tand strongly to AGG repeats (head-to-head), with asignificant requirement for orientation of this unit.Interestingly, the relative sizes and mobility ofLEDGF complexes with DNAs containing varioussequences divergent from its core binding elementwere almost identical, based on their electro-phoretic mobility on native gels (Figure 9). Theseresults suggest LEDGF binds as a monomer undernormal physiological conditions. We consider thatthe stability and specificity of LEDGF–DNA inter-actions may be separately achieved by N andC-terminal domains, respectively. It has beenreported that dimeric proteins can bind with dyadsymmetry to palindromic DNA sequenceelements,53,54 GCN4 to its upstream activationsequence (UAS),55 and estrogen receptor to the

estrogen response element (ERE).56 Alternatively,repetitive structures within a single polypeptidecan interact with tandem repeats of short sequenceas in the case of TFIIIA binding to the internalcontrol region of the 5 S RNA gene.57 Our datademonstrate that LEDGF’s interaction with itselements does not fall into either of these examples.We believe that LEDGF has the ability to form stablecomplexes with nAGGn and nGAAn units. Suchremarkable flexibility in protein DNA interaction isindeed remarkable, and this may be a generalproperty of LEDGF, namely that the N-terminalprovides stability and the C-terminal domainprovides specificity. We suggest that a proteinhaving such different distinct domains may wellhave different functions, some of which areinvolved in the stabilization of binding. Moreover,there is the possibility of interaction(s) of its domainwith other proteins, since LEDGF has recently beenimplicated in protein–protein interactions, genesilencing and regulation by binding to chromatinand transcriptional regulation.37,40,41,58–60 Theidentification of these putative unknown proteinsand DNA elements recognized by LEDGF wouldgreatly help to further elucidate both the physio-logical role and effective mechanism of thismolecule in terms of DNA binding of proteinsalong the proteome and genome during develop-ment and cellular homeostasis.

Materials and Methods

Cell culture and transfection

Cos-7 cells, LECs, and fibroblasts were cultured inDulbecco’s Modified Eagle’s Medium (DMEM; Gibco-BRL Life Technologies) supplemented with 10% (v/v)fetal bovine serum (FBS; Gibco-BRL) in 75 mm tissueculture flasks in a 5% (v/v) CO2 environment at 37 8Cfollowing standard procedures. Cells were trypsinized(0.25%) and plated in culture dishes at a density of 5!105

in 60 mm plates or 1!106 in 100 mm plates whenrequired. All transfection procedures were performedusing Superfectamine (Qiagen). To assess the effect ofLEDGF and its N and C-terminal domains, cells weretransfected with LEDGF, NI-LEDGF, and CI-LEDGFlinked to EGFP vector. Cells were grown in DMEM with10% (v/v) FBS, and selected as described.27 These cellswere used to monitor the ability of each protein inpromoting cellular survival against paraquat-inducedoxidative stress.28

Plasmid construction, expression, and purification offull-length LEDGF and truncated GST-LEDGF protein

Subcloning techniques described by Sambrook et al.61

were used throughout these experiments. A fusion proteinbetween LEDGF4 and partial LEDGF with glutathione-S-transferase (GST), generated by inserting the entire codingsequence of the LEDGF cDNA into the BamHI and EcoRIsites of a pGEX-2T vector (Pharmacia Biotech, Piscataway,NJ), was used to transform Escherichia coli (BL21).16 Theexpression of the GST-LEDGF fusion protein was inducedwith isopropyl-D-thiogalactopyranoside (IPTG). Proteinswere purified with glutathione-Sepharose 4B beads


(Pharmacia Biotech) following the manufacturer’s proto-cols. The protein concentration was determined with theBradford method.62

Cloning of GFP-LEDGF and truncated GFP-LEDGF toascertain its sub-cellular localization by fluorescentmicroscopy

GFP-tagged LEDGF was made using the pEGFP(Clontech), which allows LEDGF fusion to the C terminusof GFP. Primers corresponding to the N and C-terminalcoding regions of LEDGF were tagged with BglII andEcoRI sites. This construct was used in the PCR reactionas the cDNA template to generate different constructs.PCR product corresponding to destined regions wascloned in TOPO-GFP vector (Strategene) to allow an in-frame fusion (GFP-LEDGF). The sizes of variousconstructs were as follows: LEDGF-Full (aa 1–530),LEDGF-NI (aa 1–187), LEDGF-NII (aa 1–135), LEDGF-NIII (aa 1–101), LEDGF-NIV (aa 1–68), LEDGF-NV (aa1–35), LEDGF-CI (aa 199–530), LEDGF-CII (aa 302–530),LEDGF-CIII (aa 322–530), LEDGF-CIV (aa 348–530),LEDGF-CV (aa 418–530), LEDGF-CVI (aa 443–530).These GFP-tagged constructs were transfected intothree cell lines (hLECs, Cos7, and fibroblasts), andsubcellular localization was directly visualized witha Nikon-inverted microscope. However, we havepresented results of Cos7 cells only. Similar constructswere made using a prokaryotic vector, pGEX2T(Amersham Pharmacia Biotech).

The following primers were used for PCR amplificationto generate the various constructs:

LEDGF-NIfor:5 0-CCCCGGATCCATGACTCGCGATTTCAAACCT-3 0

LEDGF-NIrev:5 0-TCTTGAATTCTGTAGCTGCAGGTCGTCCTCT-3 0

LEDGF-NIIrev:5 0-CTTCCTTTGGAATTCTAGTTTGG-3 0

LEDGF-NIIIrev:5 0-TCATCAGATGATGAATTCGATTG-3 0

LEDGF-NIVrev:5 0-ATGAATTCGTGGCTTTACAGCTCC-3 0

LEDGF-NVrev:5 0-CATCAGATGATGAATTCGATTG-3 0

LEDGF-NVIrev:5 0-CCGAATTCTTTTATCCGGCTCTTTTCTTGGC-3 0

LEDGF-CIfor:5 0-ATGGTAAAACAGCCCTGTCCTTC-3 0

LEDGF-CIIfor:5 0-ATGCTGAAAGGCCAACATGAC-3 0

LEDGF-CIIIfor:5 0-ATGGAAACTGAGCAGCAGAATAAAG

LEDGF-CIVfor:5 0-ATGGATTCTCGACTTCAAAGGATAC-3 0

LEDGF-CVfor:5 0-ATGGAAAAGTCTACAATGTTG-3 0

LEDGF-CVIfor:5 0-GAATGGATCCCTTGCTGAACAAAGACAGC-3 0

LEDGF-CVIIfor:5 0-CAGGATCCGCATCTGTTAATCTA-3 0

LEDGF-C�rev:

5 0-TCCAGGTATGTCAACCTAG-3 0

Constructs having AT-hook domain

To explore the role of AT-hook-like domain of LEDGFin DNA binding, we engineered LEDGF-NVI (aa 1–250;

Figure 1) as well as LEDGF-CVII (aa 170–530; Figure 1)constructs of LEDGF containing AT-hook-like domainusing primers (LEDGF-NVIrev and LEDGF-CVIIfor).Protein was purified and DNA activity was assessed.

In vitro mutagenesis

PCR-based site-directed mutagenesis was performedusing the Quickchangee Site-Directed Mutagenesis kit(Stratagene) and following the company’s protocol.Briefly, amino acid exchanges were generated by pointmutations in the GFP-LEDGF-NI containing putativeNLS (146-RRGRKRKAEKQ-156) cDNA using thefollowing complimentary primers (changed nucleotidesare underlined):

MU145: 50-GCT GCC ATA AGG GGG ATA AAG ATA AAG GCAGAA AAA CAA G-3 0 and 5 0-C TTG TTT TTC TGC CTT TATCTT TAT CCC CCT TAT GGC AGC-3 0

MU147: 5 0-GCC AGA AGG GGG ATA AAG ATA AAG GCA GAAATA CAA GTA G-3 0 and 5 0-C TAC TTG TAT TTC TGC CTTTAT CTT TAT CCC CCT TCT GGC-3 0

Following site-directed mutagenesis, Epicuran ColiXL2-Blue super-competent cells (Stratagene) were sub-sequently transformed with mutated cDNAs, and cloneswere grown on Luria-Bertani/Kan Petri dishes. Theplasmid was amplified, and the mutation was confirmedby sequencing. During the SDM experiments, the primer,MU145, failed to create a point mutation at the destinedamino acids; thus it could mutate only at two positions(Mut1, Figure 8(b)). The Mut1 plasmid was further usedas a template to create Mut2 using the same primers(Figure 8(b)).These mutated plasmids were transfectedand intracellular translocation was determined withfluorescent microscopy.

Western blot analysis

Whole cell extracts were prepared as described,16

and protein blot analysis was conducted. Equalamounts of protein samples were loaded onto a SDS-10% (w/v) PAGE, blotted onto a PVDF membrane(Immobilone-P; Millipore Corporation, Bedford, MA),and then immunostained with primary antibodies atappropriate dilutions (anti-LEDGF antibody or GSTantibodies). The filters were then incubated withhorseradish peroxidase conjugated secondary anti-bodies. The specific protein band was visualized byincubating the membrane with luminol reagent (SantaCruz Biotechnology) and exposing the membrane tofilm (X-OMAT; Eastman Kodak).

Affinity chromatography

Affinity chromatography was carried out following themethod described.10 Briefly, a double-stranded random-ized 12-mer oligo 62 was radiolabeled63 and used for thestudy. The binding site selection was performedfollowing the method of Oliphants et al.64 and Jonget al.58 Purified N-terminal LEDGF was covalently boundto CNBr-activated Sepharose 4B (Pharmacia) in couplingbuffer containing 0.1 M NaHCO3 (pH 8.3) and 0.5 MNaCl. The remaining active groups were deactivatedby 0.1 M Tris–HCl (pH 8.0) according to the manufac-turer’s instructions. The oligo 62 probe was suspendedin 100 ml of buffer C (50 mM Tris (pH 8.0), 1 mM DTT,10 mg/ml (w/v) of gelatin, 0.1 M NaCl, 10 mg/ml of


poly(dI/dC), and 20 mg/ml of tRNA). This probe(106 cpm) was added on a micro (100 ml) LEDGF-Sepharose column. Three rounds of selection wereperformed for proper binding of DNA and protein. Thecolumn was washed with buffer C containing 100 mMNaCl to remove loosely bound probe. The bound oligoswere eluted with high concentrations of salt (1 M NaCl).These oligos were amplified by PCR and ligated to abluescript vector using the EcoRI and BamHI sites, andwere sub-cloned and sequenced.

Electrophoretic mobility shift assay (EMSA)

EMSAwas performed as described elsewhere.10,16 Oligoswere commercially synthesized, annealed, and end-labeledwith [g-32P]ATP using T4 polynucleotide kinase (NewEngland Biolabs, Inc). The binding reaction was performedin 20 ml of binding buffer (20 mM Tris–HCl (pH 8.0), 75 mMKCl, 5% (v/v) of glycerol, 50 mg/ml of bovine serumalbumin, 0.025% (v/v) of NP-40, 1 mM EDTA, 5 mM DTT,and 1 mg of poly(dI/dC). Then 5 fmol of end-labeled probewas incubated with recombinant GST-LEDGF fusionprotein on ice for 30 min. The samples were then loadedon 5% or 6% polyacrylamide gel in 0.5! TBE buffer for2 h at 10 V/cm.27 The gel was dried and autoradiographed.In a competition assay, 1000-fold molar excess of coldprobe was added. For the supershift assay, 1 ml ofrabbit-anti-LEDGF Ab was added to the binding reaction,and this was further incubated for 30 min.

Construction of Hsp27 gene promoter linked to CAT

The Hsp27-CAT construct was engineered asdescribed.10 Briefly, the 5 0-flanking region of the humanHsp27 gene was isolated with a genomic PCR kit(Clontech) using specific primers. A forward primercontaining a SacI site (5 0-GCGTCGAGCTCTCGAATTCATTTGCTT-3 0) and reverse primer with an XhoI site(5 0-GCTCTCGAGGTCTGCTCAGAAAAGTGC-3 0) were used togenerate the fragment, which was cloned between theEcoRI sites of the TA vector (Invitrogen Corp., Carlsbad,CA). The Hsp27/TA construct was digested with SacI andXhoI (K214 to C21), and the promoter fragment wasligated to a pCAT-Basic vector (Promega), using theappropriate restriction enzymes.

Transfection and chloramphenicol acetyltransferaseassay (CAT assay)

The CAT assay was performed using a CAT-ELISA kit(Roche Diagnostics GmbH, Germany). Cells weretransfected/co-transfected with Superfectamine withHsp27/CAT reporter construct and/or pEGFP expressionvector, and 1 mg of SEAP vector.17 After 72 h of incubation,cells were harvested, and extracts were prepared andnormalized. CAT-ELISA was performed to monitorCAT activity, following the manufacturer’s protocol.Absorbance was measured at 405 nm using a microtiterplate ELISA reader. Trans-activation activities wereadjusted for transfection efficiencies using SEAP values.17

Acknowledgements

The authors gratefully acknowledge financialsupport from the National Eye Institute, NIH

(RO1 13394 to D.P.S.) and Research for PreventingBlindness (to R.P.B.).

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Edited by M. Yaniv

(Received 30 April 2005; received in revised form 15 October 2005; accepted 19 October 2005)Available online 9 November 2005

DNA Binding Domains and Nuclear Localization Signal of LEDGF: Contribution of two Helix-Turn-Helix...

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