Krüppel Like Factor 4 Promoter Undergoes Active Demethylation during Monocyte/Macrophage Differentiation

The role of different lineage specific transcription factors in directing hematopoietic cell fate towards myeloid lineage is well established but the status of epigenetic modifications has not been defined during this important developmental process. We used non proliferating, PU.1 inducible myeloid progenitor cells and differentiating bone marrow derived macrophages to study the PU.1 dependent KLF4 transcriptional regulation and its promoter demethylation during monocyte/macrophage differentiation. Expression of KLF4 was regulated by active demethylation of its promoter and PU.1 specifically bound to KLF4 promoter oligo harboring the PU.1 consensus sequence. Methylation specific quantitative PCR and Bisulfite sequencing indicated demethylation of CpG residues most proximal to the transcription start site of KLF4 promoter. Cloned KLF4 promoter in pGL3 Luciferase and CpG free pcpgf-bas vectors showed accentuated reporter activity when co-transfected with the PU.1 expression vector. In vitro methylation of both KLF4 promoter oligo and cloned KLF4 promoter vectors showed attenuated in vitro DNA binding activity and Luciferase/mouse Alkaline phosphotase reporter activity indicating the negative influence of KLF4 promoter methylation on PU.1 binding. The Cytosine deaminase, Activation Induced Cytidine Deaminase (AICDA) was found to be critical for KLF4 promoter demethylation. More importantly, knock down of AICDA resulted in blockade of KLF4 promoter demethylation, decreased F4/80 expression and other phenotypic characters of macrophage differentiation. Our data proves that AICDA mediated active demethylation of the KLF4 promoter is necessary for transcriptional regulation of KLF4 by PU.1 during monocyte/macrophage differentiation.


Introduction
Myeloid cell differentiation is controlled by a complex circuitry of lineage specific transcription factors and the role of the Ets family transcription factor PU.1 in myeloid lineage specification is well documented [1,2]. PU.1 expression levels critically determine the specification of myeloid and common lymphoid progenitors [3] and knock down of PU.1 in mice results in defective development of macrophages and granulocytes [4,5]. Interestingly, high levels of PU.1 support macrophage development whereas low levels support the production of granulocytes [6]. Also, cooperative or antagonistic interactions of PU.1 with other transcriptional factors decides the cell fate towards erythrocytic, B-cell, mast or dendritic cell lineages [7,8]. The importance of Krüppel like factor 4 (KLF4) in inflammatory monocyte differentiation in vivo and also in early monocyte development was identified by Alder et al [9] and Feinberg et al [10][11]. X-ray crystal studies of KLF4 protein revealed that the deletion of the two C-terminal zinc fingers lead to deficiency of KLF4 expression resulting in macrophage self renewal and defective differentiation [12]. Recent genome wide studies on epigenetic and transcription factor profiling in different cell types correlated the lineage specific transcription factor binding with changes in epigenetic marks like histone acetylation/methylation and DNA methylation at the target gene promoters that potentially affect the cell fate.
DNA methylation is a dynamic epigenetic change that regulates gene expression by controlling accessibility of DNA to transcription factor binding. DNA methylation marks are lost either by passive or active mechanisms where passive demethylation occurs in actively replicating cells and active demethylation is observed in non replicating cells. The role of mammalian DNA methylating enzymes DNA methyl transferase 3A/3B and DNMT1 in maintaining DNA metjhylation marks is well characterized whereas the mechanisms of demethylation have been elusive and it was believed that it occurs by a passive process during DNA replication. The process of active demethylation and exact mechanism is still under debate [13][14][15][16]. Initial studies reported that active DNA demethylation to be associated with DNA Base Excision Repair proteins like Thymine DNA Glycosylase (TDG), Methyl CpG binding Domain (MBD) and Growth Arrest and DNA Damage inducible (GADD45) proteins [17][18][19][20]. Activation Induced Cytidine Deaminase (AICDA) that deaminates methyl Cytosine was also reported to play a crucial role in active DNA demethylation [21][22][23]. Recent studies also advocated coupled action of GADD45a and TDG or Ten Eleven Translocation (TET) proteins followed by the deaminase AICDA for active demethylation [19,20]. Data on generation of 5 methyl cytosine intermediates like 5 hydroxylmethyl cytosine, 5 formyl cytosine, 5 carboxyl cytosine that are further calalyzed by TDG via base excision repair or TET proteins indicate additional levels of regulation during the active demethylation [24][25][26][27]. Another interesting finding is that, the deaminase AICDA also alters subcellular localization of TET family proteins, which may possibly affect the TET mediated demethylation [28]. Despite the extensive efforts, direct evidence on the role of these protein factors in 5 mC base conversions is still incomplete and knock out mice studies are still inconclusive [21,29]. In our studies to delineate the epigenetic mechanisms involved in PU.1/KLF4 dependent monocytic macrophage differentiation, we observed that expression of KLF4 is regulated by PU.1 at transcriptional level and this event is linked with active demethylation of KLF4 promoter. In the present study using non-proliferating PU.1 inducible myeloid progenitor cells and differentiating bone marrow derived macrophages (BMDM), we aimed to identify the regulation of KLF4 promoter demethylation during monocyte/macrophage differentiation and the mechanism/protein factor(s) involved in this process.

Materials and Methods
Cells PU/ER(T) cell line was a gift from Dr. Harinder Singh, University of Chicago [30]. PU/ER(T) cells were grown in IMDM supplemented with 1% Glutamine, 1% b-mercapto ethanol, 10%FBS, 50 IU/mL Penicillin and 50 mg/mL Streptomycin and 5 ng/mL IL-3. Bone marrow cells were differentiated in to mature macrophages (BMDMs) from wild type and GADD45a mice in DMEM supplemented with 10% FBS, 50 IU/mL Penicillin and 50 mg/mL Streptomycin and 20 ng/mL recombinant mouse M-CSF added on Day 1, 3 and 5. Cells were allowed to differentiate up to 7 days in total. HEK 293 cells were cultured in DMEM F12 with 10% FBS and 50 IU/mL Penicillin and 50 mg/mL Streptomycin.

Mice
Wild type C57 black6 mice were purchased from Jackson Laboratories. GADD45a knock out mice were a gift from Dr Jeffery Jacobson at the Institute of Personalized Respiratory Medicine, University of Illinois Chicago. All the animal experiments and procedures were conducted with protocols approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Illinois at Chicago and The Ohio State University, Columbus.

Western Blot Analysis
PU/ER(T) cells with and without Tamoxifen treatment or bone marrow derived macrophages from wild type or GADD45a knock out mice were harvested, and cell extracts were prepared in 1X RIPA buffer (Cell signaling Technologies #9806) supplemented with protease inhibitors. An equal amount of protein was analyzed by Western blotting for the protein of interest using its specific antibodies according to standard protocols.

RT-PCR
Total cellular RNA was isolated from cells under different experimental conditions using QIAGEN RNeasy Plus Mini Kit (#74134) following the manufacturer's instructions. Reverse transcription was carried out with the Fermentas cDNA synthesis kit for RT-PCR using the supplier's protocol, and the cDNA was then used as template for PCR using primers (listed in Supplement S1, at least one of the primer was picked from exon/exon junction for mRNA expression assay) specific for mouse KLF4, GATA1, GATA2, AICDA and b-Actin. The PCR amplification was carried out using Applied Biosystems SYBR green reaction mix (#4309155) on Applied Biosystems Real Time PCR machine 7500 or Roche 480 PCR machine.

Electrophoretic Mobility Shift Assay
Nuclear extracts of Tamoxifen treated PU/ER(T) or bone marrow derived macrophages during different differentiation stages were prepared and analyzed for DNA binding activity. 59 biotin labeled KLF4 promoter oligo was incubated with 5 mg nuclear protein extract, resolved on 6% native Acrylamide gels using 0.5X TBE buffer. The protein DNA complexes from the gels were transferred on to Biodyne A Nylon membrane (#77015) and developed using North2South Chemiluminescent Developing reagent kit (#89880) from Pierce according to the supplier's instructions.
Chromatin Immunoprecipitation (ChIP) Assay PU/ER(T) or BMDM cells were subjected to appropriate treatments, cross linked using 1% formaldehyde for 10 min at 37uC and washed with PBS. Cross linking was stopped with 0.125 M glycine in PBS, cells were washed in PBS, centrifuged for 5 min at 1200 rpm, and pellets were processed using the buffers supplied in QIAGEN Epitect one day Chromatin Immunoprecipitation kit. Cell pellets were sonicated and pre-cleared using Protein A agarose supplied in the kit. Resulting Chromatin was Immunoprecipitated using Chip grade PU.1 or AICDA antibodies and respective pre-immune serum. 1% of pre-cleared chromatin was set aside as input control, de-cross linked and processed along with the immunoprecipitated fractions before subjecting to real time PCR. Quantitative PCR results were calculated using the SABioscience EpiTecChIP q PCR Data Analysis Template (http://www.sabiosciences.com/chippcrarray_data_analysis.php) and presented as % Input values.

Cloning of mouse KLF4 Promoter
The mouse KLF4 promoter region 21481 to +45 bp relative to the transcription start site was PCR-amplified from genomic DNA using primers listed in Supplement S1. PCR product was analyzed by agarose gel electrophoresis and cloned into the pcr 2.1 TOPO vector at SacI and EcoRV. The PCR insert was released by digestion with SacI and SmaI and subcloned in to pGL3 yielding pGL3-KLF4(1.6 kb)-Luciferase. KLF4 insert was released from pGL3 using SpeI, HindIII and subcloned in to pcpgf-bas vector [31]. Nucleotide sequence of each construct was verified by DNA sequencing.

Transient Transfection and Luciferase/mouse Alkaline Phosphotase (mSEAP) reporter Assays
An equal number (5-6610 6 ) of PU/ER(T) or HEK293 cells were electroporated with pGL3-KLF4 promoter constructs and pRLTK using Amaxa Electroporation kit. 36 h after transfection, cells were washed with cold PBS and lysed in 100 ml of Promega cell lysis buffer. Cell extracts were assayed for luciferase activity using a Promega dual luciferase assay system. Similarly, HEK293 cells were electroporated with pcpgf-KLF4 and grown in Invivogen HEK-Blue detection medium. mSEAP reporter activity was measured by absorbance of the growth medium at 630 nm.
Bisulfite conversion, methylation specific PCR and sequencing. Total genomic DNA from PU/ER(T) or wild type and GADD45a-/-bone marrow derived macrophages at different stages of differentiation was isolated and treated with MethylCode Bisulfite conversion reagent. The bisulfite converted DNA was purified and sequenced using M1CpG specific primers used for methylation specific PCR, listed in Supplement S1. DNA sequence results were subjected to multiple alignment using BiQ Analyzer [32], CpG dinucleotide distribution was plotted as bubble chart with filled circles representing methylated cytosine and open circles with demethylated cytosine bases.
Methylation Specific PCR. Methylation Specific Primers for KLF4 and GATA2 Promoters were designed using MethPrimer program [33]. Bisulfite converted genomic DNA was PCR amplified using methylation specific primers and SYBR green reaction mix. The methylation index was calculated as the ratio of methylated DNA cp values to the sum of cp values of methylated DNA and unmethylated DNA. The methylated and unmethylated DNA specific primer sequences are listed in Supplement S1.

FACS analysis
Single-cell suspension of PU/ER(T) (1-2610 4 /sample) was washed and incubated on ice for 30 minutes with appropriate fluorescently labeled antibodies. Cells were analyzed on an FACS Vantage flow cytometer (BD Biosciences) where gating was set based on respective unstained cell population and isotype matching control staining. The data were analyzed with FlowJo software.

Statistical analysis
All the experiments were repeated three times for consistent results and representative data sets for EMSA, WB and flow cytometry are presented. Statistical analyses were done using GraphPad-Instat software with Mann-Whitney U test and significance in differences is calculated. p#0.05 was considered significant. Error bars represent Standard Deviation.

PU.1 specifically binds to KLF4 promoter
We used PU/ER(T) cell line generated from fetal liver cells of PU.1-/-mice that shows conditional PU.1 dependent macrophage differentiation [30,34]. In these mice the wild type PU.1 gene is knocked in by 2 alleles of the PU/ER(T) transgene that was constructed by fusion of the modified estrogen receptor ligand binding domain-G525R, inducible by tamoxifen. In the presence of tamoxifen, the PU/ER(T) fusion protein is translocated into nucleus and becomes functionally active similar to WT PU.1, but in the absence of tamoxifen the fusion molecule remains in the cytoplasm [34]. As seen in Figure 1A, PU.1 protein translocates completely in to nucleus with in 1 h on stimulation with 100 nM of the estrogen receptor ligand 4-Hydroxy Tamoxifen. KLF4 is one of the target genes of PU.1 and to determine if the translocated nuclear PU.1 protein modulates KLF4 transcriptionally, we analyzed the promoter sequence of KLF4 for PU.1 binding elements by TF search [35]. KLF4 promoter shows two PU.1 consensus binding elements spanning from 2118 to 2113 and 240 to 235 bp upstream of transcription start site. Binding of PU.1 to its consensus sequence in KLF4 promoter was determined by Electrophoretic Mobility Shift Assay (EMSA) using a biotin labeled 76 bp KLF4 promoter oligo harboring the 2118/2113 PU.1 binding element. Nuclear protein extracts from differentiating wild type mouse BMDM and tamoxifen treated PU/ER(T) cells showed increased binding of PU.1 to KLF4 promoter oligo indicating possible transcriptional regulation of KLF4 by PU.1 (Figure 1

KLF4 promoter undergoes demethylation during monocyte/macrophage differentiation
In silico analysis of KLF4 promoter using Methprimer [33] showed dense hypermethylation with CpG dinucleotides distributed spanning the 1.6 kb region (Figure 3 A & B). As we observed that PU.1 binds to the KLF4 promotor during macrophage differentiation, methylation status of KLF4 promoter DNA is an important factor to determine its accessibility to PU.1 binding. Methylation specific quantitative PCR primers were designed separately for the three CpG regions, arbitrarily designated as M3CpG (spanning 21432 to 21321), M2CpG (spanning 2884 to 2689) and M1CpG (spanning 2627 to +73) using the MethPrimer tool [33]. Using the methylation specific primers we analyzed the methylation status of KLF4 promoter in both Tamoxifen inducible PU/ER(T) cell line and wild type BMDM during early and late differentiation stages by bisulfite sequencing and quantitative PCR. Interestingly, the M1CpG region most proximal to the KLF4 transcription start site is demethylated indicated by decreased methylation index and bisulfite sequencing during differentiation of BMDM and PU/ER(T) cells (Figure 3

PU.1 transcriptionally regulates KLF4 promoter
To analyze the effect of PU.1 on transcriptional regulation of KLF4 promoter, we cloned 1.6 kb of KLF4 promoter in to pGL3 Luciferase promoter probe vector and CpG free mSEAP reporter  was determined using methylation specific primers designed using Methprimer. G) PU/ER(T) cells were grown in presence of 95% Ethanol or 100 nM Tamoxifen for 24 h and total cellular RNA was isolated and reverse transcribed. Using the resulting cDNA as template relative expression levels of KLF4, GATA1 and GATA2 normalized to b-Actin were determined by gene specific primers and SYBR green reaction mix. Error bars represent standard deviation. Figure

PU.1 Binding is specific to demethylated KLF4 promoter
To determine the effect of KLF4 promoter methylation on PU.1 binding, we in vitro methylated the CpG dinucleotides in the biotin labeled 76 bp KLF4 promoter oligo sequence by M. SssI in presence of S-Adenosyl Methionine and used as a probe in DNA binding assay. Tamoxifen treatment increased PU.1 binding to KLF4 promoter whereas this binding was completely abolished when the KLF4 promoter was in vitro methylated (Figure 4 F, lanes 3 to 5 versus 8 to 10). In vitro methylation of KLF4-pGL3 promoter probe vector by M. SssI completely abolished the KLF4 promoter reporter gene activity (Figure 4 G). The contribution of CpG residues in pGL3 vector back bone during in vitro methylation experiment was ruled out by using KLF4 promoter cloned in pcpgf-bas (Invivogen), CpG free mSEAP reporter vector with no CpG dinucleotides in the pGL3 vector back bone [31]. pcpgf-KLF4 construct also showed similar attenuation of promoter activity when in vitro methylated (Figure 4 H). This data consistently proved that demethylation of KLF4 promoter is essential for effective binding of PU.1 and subsequent transcriptional regulation of KLF4.

Expression of Active demethylation proteins in PU/ER(T) cells
Next, we aimed to identify the mechanism of KLF4 promoter demethylation during monocyte/macrophage differentiation and if the proteins AICDA, GADD45a, MBD4, TDG or TET2 are involved in this process. Expression and localization of these proteins in cytosolic and nuclear compartments was determined by western blotting in differentiating PU/ER(T) cells treated with 100 nM tamoxifen. AICDA, GADD45a, MBD4, TDG or TET2 were expressed constitutively in PU/ER(T) cells and are localized predominantly in the nuclear fraction of tamoxifen treated PU/ ER(T) cells (Figure 5 A). As AICDA is the proposed deaminase in GADD45a or TET mediated demethylation mechanisms, we investigated the role of AICDA in active demethylation of KLF4 promoter. Interestingly, AICDA was extensively studied in B-cells and its expression in myeloid cells was not reported. Therefore, AICDA mRNA expression was measured by quantitative RT-PCR in Tamoxifen treated PU/ER(T) cells and the RT-PCR product was further purified, sequenced and confirmed to be that of AICDA transcript (Supplement S1-H, NCBI nucleotide blast alignment).

AICDA interacts with GADD45a and TDG
To test if AICDA interacts with the other putative demethylation proteins, we performed co-Immuno precipitation experiments. Myc tagged AICDA plasmid was over expressed by electroporation in PU/ER(T) cells and total cellular protein was immunoprecipitated with Anti-myc followed by immunoblotting for GADD45a, TDG, MBD4 and TET2 proteins separately. AICDA was found to interact with GADD45a strongly followed by TDG (Figure 5 C). MBD4 and TET2 were not detected in the AICDA immunoprecipitate (data not shown). Over expression of AICDA was confirmed by immunoblotting with AICDA and Myc antibodies (Figure 5 B).
AICDA might physically interact with KLF4 promoter DNA in the chromatin and this physical interaction may be direct or indirect involving protein factors implicated in demethylation process. The physical association of AICDA to the KLF4 promoter region was assessed by recruitment of AICDA on to the KLF4 promoter by Chromatin Immunoprecipitation (ChIP) assay using AICDA antibodies in differentiating BMDM and Tamoxifen treated PU/ER(T) cells ( Figure 5 D & E). In both the cell types AICDA showed constitutive occupancy on KLF4 promoter M1CpG island region.

AICDA is essential for KLF4 demethylation
Next, we determined the KLF4 promoter M1CpG region demethylation in differentiating GADD45a-/-and wild type bone marrow cells by methylation specific qPCR and bisulfite sequencing. Interestingly, in GADD45a-/-BMDM the KLF4 promoter was demethylated similar to wild type BMDM during differentiation, not supporting the requirement of GADD45a in KLF4 promoter sequence specific demethylation (Figure 6 A). Further, we investigated if AICDA deletion alters KLF4 promoter methylation in PU/ER(T) cells by using AICDA shRNA. PU/ ER(T) cells were electroporated either with non specific control or AICDA shRNA plasmid (Origene). After 72 h of transfection the expression levels of AICDA were found to be reduced by more than 90% in AICDA shRNA transfected cells compared to control shRNA transfected cells (Figure 6

Discussion
The critical role of PU.1 and KLF4 in monocyte/macrophage differentiation is well established [9][10][11][12]. In the present study, we addressed the question if PU.1 has any effect on chromatin status in terms of methylation changes during transcriptional activation of KLF4. The significant findings of the present study can be summarized as: 1) monocyte/macrophage lineage commitment of pluripotent fetal liver cells and hematopoietic cells is determined by PU.1 dependent KLF4 expression. 2) PU.1 binds to its consensus sequence in KLF4 promoter and this binding is associated with demethylation of KLF4 promoter. 3) Cloned KLF4 promoter Luciferase/mSEAP reporter vector showed increased Luciferase/alkaline phosphotase reporter activity in presence of PU.1 expression vector 4) PU.1 failed to bind In vitro methylated KLF4 promoter oligo and also showed attenuated KLF4 promoter reporter activity when it was in vitro methylated. 5) AICDA interacts with GADD45a and TDG but GADD45a was found to be dispensable for KLF4 promoter demethylation. 6) shRNA knock down of AICDA blocked demethylation of KLF4 promoter, F4/80 expression and macrophage differentiation. Together, these observations provide a mechanistic evidence for the role of AICDA in active demethylation/regulation of KLF4 expression during monocyte/macrophage lineage commitment and differentiation.
In the current study, using PU.1 inducible cells as monocyte/ macrophage differentiation model, transcriptional regulation of KLF4 that is tightly controlled by its promoter methylation is established. In the same cellular system, we observed decreased expression of GATA1/GATA2 mRNA and maintenance of GATA2 promoter methylation during macrophage differentiation. The balance of PU.1 and GATA1/2 proteins supports the earlier literature, where GATA proteins are down regulated during myeloid lineage specification and their up regulation favors the cell fate towards erythroid lineage [36]. PU.1 is considered as the master regulator of different hematopoietic lineages and it regulates its target genes at multiple levels. During macrophage differentiation PU.1 associates with differentiation-associated epigenetic changes and binds to its target genes [37]. Binding of PU.1 was found to depend on its concentration, chromatin accessibility, PU.1 motif binding affinity and co-operative interaction with neighboring transcription factors [38]. This study also reported that PU.1 binding is independent of the genome wide DNA methylation where as our data identified a locus specific demethylation of KLF4 promoter which is required for PU.1 binding. Additionally, ChIP sequencing analysis during terminal differentiation of monocyte to osteoclasts, it was observed that PU.1 target genes undergo TET2 coupled demethylation and DNMT3b mediated methylation [39]. This study also showed that PU.1 interacts with both DNMT3b and TET2 and its knock down in primary monocytes impairs acquisition of DNA methylation marks and expression changes; reduced association of TET2 and DNMT3b at PU.1 target genes. We identified a similar role of PU.1 in associating with locus specific demethylation of KLF4 promoter and further attempted to investigate the proteins involved in demethylation.
Existing literature reported active DNA demethylation in several cell types including neurons, T-cells and human embryonic kidney cells [40,41]. GADD45a promoted active DNA demethylation in zebra fish zygote, X. laevis oocytes and HEK293T cells [42][43][44]. However, several studies failed to substantiate the role of GADD45a in demethylation of DNA in mice [45,46]. For example, neither global nor locus-specific methylation was increased in Gadd45a-/-mice [46,29]. In the present study, we observed similar phenomenon where the KLF4 promoter in GADD45a-/-myeloid progenitors was not hyper methylated but underwent normal demethylation during monocyte/macrophage differentiation.
The role of another candidate protein of active demethylation, AICDA in DNA demethylation was initially postulated by Morgan et al [22] in inducing pluripotency in oocytes, embryonic germ cells and embryonic stem cells. Bhutani et al. [47] showed that expression of AICDA is required for promoter demethylation and induction of OCT4 and NANOG gene expression in interspecies heterokaryons of fused mouse embryonic stem cells and human fibroblasts. In another study, profiling of DNA methylation throughout the genome in wild-type and AICDA-deficient mouse Primordial Germ Cells (PGCs) revealed that AICDA-deficient PGCs were up to three times more methylated than wild-type ones [21]. Similarly, our study supported the role of AICDA in sequence specific demethylation of KLF4 promoter in differentiating myeloid progenitors. We found for the first time that AICDA was expressed in myeloid progenitor cells and found to physically associate with the KLF4 promoter. shRNA mediated suppression of AICDA resulted in blockade of maturation of myeloid progenitor cells in terms of morphological. cellular changes and F4/80 expression. Maturation of myeloid progenitors is mediated by transcriptional up regulation of KLF4 that resulted from active demethylation of its promoter by AICDA. To test if GADD45a was necessary for the sequence specific demethylation of KLF4 promoter, we compared the methylation of KLF4 promoter Figure 6. AICDA is essential for KLF4 promoter demethylation. A) The relative methylation index of the proximal M1 CpG region in KLF4 promoter was compared on 1 st and 7 th day of differentiating BMDM from wild type and GADD45a knock out macrophages as described in Figure 3 C&D. B) Knockdown of AICDA protein in AICDA-shRNA or control-shRNA electroporated PU/ER(T) cells. C) Methylation index of the proximal KLF4 promoter M1 CpG region was compared in AICDA shRNA or control shRNA electroporated PU/ER(T) cells as described in Figure 3C&D. D) Expression of F4/80 was analyzed in control shRNA or AICDA-shRNA electroporated PU/ER(T) cells treated with or without tamoxifen for 72 h. E) Morphological changes in ethanol/tamoxifen treated PU/ER(T) cells were analyzed in AICDA depleted cells in comparison with control cells. Representative histograms of flow cytometry, western blots and cell staining were presented. Figure 6A and C, * is used where ever p value is #0.05 and ** is used when p#0.005. Error bars represent standard deviation. doi:10.1371/journal.pone.0093362.g006 M1CpG region in differentiating macrophages from wild type and gadd45a knock out BMDM by methylation specific PCR. Interestingly, the gadd45a deficient macrophages did not show any defect in demethylation of the KLF4 promoter sequence, where as knock down of AICDA by shRNA drastically impaired the demethylation of KLF4 promoter and the morphological characters. In the present study, we observed a close correlation between demethylation of proximal CpG dinucleotides in KLF4 gene promoter, PU.1 binding, regulation of KLF4 expression and monocyte/macrophage differentiation. Our study postulated an important role of PU.1 binding to KLF4 promoter that depends on PU.1 binding affinity, and chromatin accessibility by changes in methylation status of KLF4 promoter DNA. Further investigations are required to determine the exact role of these site-specific methylation changes and other epigenetic modifications like histone methylation and or acetylation on macrophage differentiation. In summary, KLF4 promoter undergoes active DNA demethylation during monocyte/macrophage differentiation and AICDA plays an essential role in this demethylation process.

Supporting Information
Supplement S1 Supplement S1 contains primer sequences used for A) methylation specific qPCR B) Primers used for sequencing bisulfite converted genomic DNA C) Primers used for methylation specific PCR of GATA2 pomoter D) 59 biotin labelled 76 bp KLF4 promoter oligo used for EMSA E) Primers used for cloning KLF4 promoter F) Primers used for KLF4 expression G) Primers used for AICDA expression H) Sequencing and confirmation of AICDA transcript from differentiating PU/ER(T) cells.