The INO80 chromatin remodeling complex is involved in the nucleotide excision repair pathway

The genomic DNA of eukaryotic cells is well organized into chromatin structures. However, these repressed structures present barriers that block the access of regulatory factors to the genome during various nuclear events. To overcome the obstacle, two major cellular processes, post-modification of histone tails and ATP-dependent chromatin remodeling, are involved in reconfiguring chromatin structure and creating accessible DNA. Despite the current research progress, much remains to be explored concerning the relationship between chromatin remodeling and DNA repair. Recently, one member of the ATP-dependent chromatin remodeling complexes, INO80, has been found to play a crucial role in DNA damage repair. However, the functions of this complex in higher eukaryotes have yet to be determined. The goal of my study is to generate a human somatic INO80 conditional knockout model and investigate the functions of Ino80 in damage repair.^ By homologous targeting of the INO80 locus in human HCT116 colon epithelial cells, I established a human somatic INO80 conditional knockout model. I have demonstrated that the conditional INO80 cells exhibited a sufficiently viable period when the INO80 protein is removed. Moreover, I found that loss of INO80 resulted in deficient UV lesion repair in response to UV while the protein levels of the NER factors such as XPC, XPA, XPD were not affected. And in vitro repair synthesis assay showed that the NER incision and repair synthesis activities were intact in the absence of INO80. Examination on the damage recognition factor XPC showed its recruitment to damage sites was impaired in the INO80 mutant cells. Loss of INO80 also led to reduced enrichment of XPA at the site of UV lesions. Despite the reduced recruitment of XPC and XPA observed in INO80 mutants, no direct interaction was detected. Meanwhile, direct interaction between INO80 and DDB1, the initial UV lesion detector, was detected by coimmunoprecipitation. UV-induced chromosome relaxation was reduced in cells devoid of INO80. These results demonstrate the INO80 complex may participates in the NER by interacting with DDB1 and having a critical role of in creating DNA accessibility for the nucleotide excision pathway. ^

XENOESTROGEN-SPECIFIC MECHANISMS OF DEVELOPMENTAL REPROGRAMMING CORRELATE WITH GENE EXPRESSION AND TUMOR DEVELOPMENT

Environmental exposures during sensitive windows of development can reprogram normal physiological responses and alter disease susceptibility later in life in a process known as developmental reprogramming. We have shown that neonatal exposure to the xenoestrogen diethylstilbestrol (DES) can developmentally reprogram the reproductive tract in genetically susceptible Eker rats giving rise to complete penetrance of uterine leiomyoma. Based on this, we hypothesized that xenoestrogens, including genistein (GEN) and bisphenol A (BPA), reprogram estrogen-responsive gene expression in the myometrium and promote the development of uterine leiomyoma. We proposed the mechanism that is responsible for the developmental reprogramming of gene expression was through estrogen (E2)/ xenoestrogen inducedrapid ER signaling, which modifies the histone methyltransferase Enhancer of Zeste homolog 2 (EZH2) via activation of the PI3K/AKT pathway. We further hypothesized that there is a xenostrogen-specific effect on this pathway altering patterns of histone modification, DNA methylation and gene expression. In addition to our novel finding that E2/DES-induced phosphorylation of EZH2 by AKT reduces the levels of H3K27me3 in vitro and in vivo, this work demonstrates in vivo that a brief neonatal exposure to GEN, in contrast to BPA, activates the PI3K/AKT pathway to regulate EZH2 and decreases H3K27me3 levels in the neonatal uterus. Given that H3K27me3 is a repressive mark that has been shown to result in DNA methylation and gene silencing we investigated the methylation of developmentally reprogrammed genes. In support of this evidence, we show that neonatal DES exposure in comparison to VEH, leads to hypomethylation of the promoter of a developmentally reprogrammed gene, Gria2, that become hyper-responsive to estrogen in the adult myometrium indicating vi that DES exposure alter gene expression via chromatin remodeling and loss of DNA methylation. In the adult uterus, GEN and BPA exposure developmentally reprogrammed expression of estrogen-responsive genes in a manner opposite of one another, correlating with our previous data. Furthermore, the ability of GEN and BPA to developmental reprogram gene expression correlated with tumor incidence and multiplicity. These data show that xenoestrogens have unique effects on the activation of non-genomic signaling in the developing uterus that promotes epigenetic and genetic alterations, which are predictive of developmental reprogramming and correlate with their ability to modulate hormone-dependent tumor development.

Function and regulation of the Pem homeobox gene in vivo

Pem, a member of the PEPP homeobox family, is expressed in somatic cells in male and female reproductive tissues. In the adult murine testis, Pem is specifically expressed in Sertoli cells, where it is restricted to stages IV–VIII of the seminiferous epithelial cycle. To identify Pem's function in Sertoli cells, transgenic mice were generated that express Pem in Sertoli cells during all stages of the seminiferous epithelial cycle. This resulted in an increase in double-strand DNA breaks in preleptotene spermatocytes and single-strand DNA breaks in elongating spermatids. My results suggest that Pem regulates Sertoli-cell genes that encode secreted or cell-surface proteins that serve to control premeiotic DNA replication, DNA repair, and/or chromatin remodeling in the adjacent germ cells. Three additional transgenic mouse containing varying lengths of the Pem male-specific promoter (Pp) were generated to identify the sequences responsible for regulating Pem expression in the testis and epididymis. My analysis suggests that there are at least two regulatory regions in the Pem Pp. In the testis, region II directs androgen-dependent expression specifically in Sertoli cells whereas region I fine-tunes stage-specific expression by acting as a negative regulator. In the epididymis, region II confers androgen-dependent, developmentally-regulated expression in the caput whereas region I prevents inappropriate expression in the corpus. I also report the identification and characterization of two human PEPP family members related to Pem that I have named hPEPP1 and hPEPP2. The hPEPP1 and hPEPP2 homeodomains are more closely related to PEPP subfamily homeodomains than to any other homeodomain subfamily. Both genes are localized to the specific region of the human X chromosome that shares synteny with the region on the murine X chromosome containing three PEPP homeobox genes, Pem, Psx-1, and Psx-2. hPEPP1 and hPEPP2 mRNA expression is restricted to the testis but is aberrantly expressed in tumor cells of different origins, analogous to the expression pattern of Pem but not of Psx-1 or Psx-2. Unlike all known PEPP members, neither hPEPP1 nor hPEPP2 are expressed in placenta, which suggests that the regulation of the PEPP family has undergone significant alteration since the split between hominids and rodents. ^

A systematic analysis of INO80 in DNA replication

ATP-dependent chromatin remodeling has been shown to be critical for transcription and DNA repair. However, the involvement of ATP-dependent chromatin remodeling in DNA replication remains poorly defined. Interestingly, we found that the INO80 chromatin-remodeling complex is directly involved in the DNA damage tolerance pathways activated during DNA replication. DNA damage tolerance is important for genomic stability and is controlled by formation of either mono-ubiquitinated or multi-ubiquitinated PCNA, which respectively induce error prone or error-free replication bypass of the lesions. In addition, homologous recombination (HR) mediated by the Rad51 pathway is also involved in the DNA damage tolerance pathways. ^ We found that INO80 is specifically recruited to replication origins during S phase in a genome-wide fashion. In addition, DNA combing analysis shows INO80 is required for the resumption of replication at stalled forks induced by methyl methane-sulfonate (MMS). Mechanistically, we find that INO80 is required for PCNA ubiquitination as well as for Rad51 mediated processing of replication forks after MMS treatment. Furthermore, chromatin immunoprecipitation at specific ARSs indicates INO80 is necessary for Rad18 and Rad51 recruitment to replication forks after MMS treatment. Moreover, 2D gel analysis shows INO80 is necessary to process Rad51 mediated intermediates at impeded replication forks. ^ In conclusion, our findings establish a novel role of a chromatin-remodeling complex in DNA damage tolerance pathways and suggest that chromatin remodeling is fundamentally important to ensure faithful replication of DNA and genome stability in eukaryotes. ^

Involvement of HMGB1 in the repair of DNA adducts and the responses to DNA damage in mammalian cells

High mobility group protein B1 (HMGB1) is a multifunctional protein with roles in chromatin structure, transcription, V(D)J recombination, and inflammation. HMGB1 also binds to and bends damaged DNA, but the biological consequence of this interaction is not clearly understood. We have shown previously that HMGB1 binds cooperatively with nucleotide excision repair (NER) damage recognition proteins XPA and RPA to triplex-directed psoralen DNA interstrand crosslinks (ICLs). Based on this we hypothesized that HMGB1 is enhancing the repair of DNA lesions, and through this role, is affecting DNA damage-induced mutagenesis and cell survival. Because HMGB1 is also a chromatin protein, we further hypothesized that it is acting to facilitate chromatin remodeling at the site of the DNA damage, to allow access of the repair machinery to the DNA lesion. We demonstrated here that HMGB1 could bind to triplex-directed psoralen ICLs in a complex with NER proteins XPC-RAD23B, XPA and RPA, which occurred in the presence or absence of DNA. Supporting these findings, we demonstrated that HMGB1 enhanced repair of triplex-directed psoralen ICLs (by nucleotide incorporation), as well as removal of UVC irradiation-induced DNA lesions from the genome (by radioimmunoassay). We also explored HMGB1's role in chromatin remodeling upon DNA damage. Immunoblotting demonstrated that, in contrast to HMGB1 proficient cells, cells lacking HMGB1 showed no increase in histone acetylation after UVC irradiation. Additionally, purified HMGB1 protein enhanced chromatin formation in an in vitro chromatin assembly system. However, HMGB1 also has a role in DNA repair in the absence of chromatin, as shown by measuring UVC-induced nucleotide incorporation on a naked substrate. Upon exploration of HMGB1's effect on several cellular outcomes of DNA damage, we found that mammalian cells lacking HMGB1 were hypersensitive to DNA damage induced by psoralen plus UVA irradiation or UVC radiation, showing less survival and increased mutagenesis. These results reveal a new role for HMGB1 in the error-free repair of DNA lesions in a chromosomal context. As strategies targeting HMGB1 are currently in development for treatment of sepsis and rheumatoid arthritis, our findings draw attention to potential adverse side effects of anti-HMGB1 therapy in patients with inflammatory diseases. ^

Functions of GCN5 and P /CAF acetyltransferases in mammalian development

Histone acetyltransferases are important chromatin modifiers that function as transcriptional co-activators. The identification of the transcriptional regulator GCN5 as the first nuclear histone acetyltransferase in yeast directly linked chromatin remodeling to transcriptional regulation. Although emerging evidence suggests that acetyltransferases participate in multiple cellular processes, their roles in mammalian development remain undefined. In this study, I have cloned and characterized the mouse homolog of GCN5 and a closely related protein P/CAF that interacts with p300/CBP. In contrast to yeast GCN5, but similar to P/CAF, mouse GCN5 possesses an additional N-terminal domain that confers the ability to acetylate nucleosomal histones. GCN5 and P/CAF exhibit identical substrate specificity and both interact with p300/CBP. Interestingly, expression levels of GCN5 and P/CAF display a complementary pattern in mouse embryos and in adult tissues, suggesting that they have distinct tissue or developmental stage specific roles. To define the in vivo function of GCN5 and P/CAF, I have generated mice that are nullizygous for GCN5 or P/CAF. P/CAF null mice are viable and fertile with no gross morphological defects, indicating that P/CAF is dispensable for development and p300/CBP function in vivo. In contrast, mice lacking GCN5 die between 10.5–11 days of gestation. GCN5 null mice are severely retarded but have anterior ectopic outgrowth. Molecular marker analyses reveal that early mesoderm is formed in GCN5 null mice but further differentiation into distinct mesodermal lineages is perturbed. While presomitic mesoderm and chodamesoderm are missing in GCN5 mutant mice, extraembryonic tissues and lateral mesoderm are unaffected. This is consistent with our finding that GCN5 expression is absent in the heart and extraembryonic tissues but is uniform throughout the rest of the embryo. Remarkably, GCN5 mutant mice exhibit an unusually high incidence of apoptosis in the embryonic ectoderm and mesoderm. Finally, mice doubly null for GCN5 and P/CAF die much earlier than mice harboring the GCN5 mutation alone, suggesting that P/CAF and GCN5 share some overlapping function during embryogenesis. This work is the first study to show that specific acetyltransferase is important for cell survival as well as mesoderm differentiation or maintenance during early mammalian development. ^

Retinoid -induced regression of human head and neck squamous cell carcinomas is associated with the induction of a pro -inflammatory response

Retinoids are Vitamin A derivatives that are effective chemopreventative and chemotherapeutic agents for head and neck squamous cell carcinomas (HNSCC). Despite the wide application of retinoids in cancer treatment, the mechanism by which retinoids inhibit head and neck squamous cell carcinomas is not completely understood. While in vitro models show that drugs affect cell proliferation and differentiation, in vivo models, such as tumor xenografts in nude mice drugs affect more complex parameters such as extracellular matrix formation, angiogenesis and inflammation. Therefore, we studied the effects of retinoids on the growth of the 22B HNSCC tumors using a xenograft model. In this system, retinoids had no effect on tumor cell differentiation but caused invasion of the tumor by inflammatory cells. Retinoid induced inflammation lead to tumor cell death and tumor regression. Therefore, we hypothesized that retinoids stimulated the 22B HNSCC xenografts to produce a pro-inflammatory signal such as chemokines that in turn activated host inflammatory responses. ^ We used real time quantitative RT-PCR to measure cytokine and chemokine expression in retinoid treated tumors. Treatment of tumors with an RAR-specific retinoid, LGD1550, had no effect on the expression of TNFα, IL-1α, GROα, IP-10, Rantes, MCP-1 and MIP-1α but induced IL-8 mRNA 5-fold. We further characterized the retinoid effect on IL-8 expression on the 22B HNSCC and 1483 HNSCC cells in vitro. Retinoids increased IL-8 expression and enhanced TNFα-dependent IL-8 induction. In addition, retinoids increased the basal and TNFα-dependent expression of MCP-1 but decreased the basal and TNFα dependent expression of IP-10. The effect of retinoids on IL-8 and MCP-1 expression was very rapid with increased levels of mRNA detected within 1–2 hours. This effect did not require new protein synthesis and did not result from mRNA stabilization. Both RAR and RXR ligands increased IL-8 expression whereas only RAR ligands activated MCP-1 expression. ^ We identified a functional retinoid response element in the IL-8 promoter that was located adjacent to the C/EBP-NFkB response element. TNFα treatment of the 22B cells caused rapid, transient and selective acetylation of regions of the IL-8 promoter associated with the NFkB response element. Co-treatment of the cells with retinoids plus TNF increased the acetylation of chromatin in this region without altering the kinetics of acetylation. These results demonstrate that ligand activated retinoid receptors can cooperate with NFkB in histone acetylation and chromatin remodeling. We believe that in certain HNSCC tumors this cooperation and the resulting enhancement of IL-8 expression can induce an inflammatory response that leads to tumor regression. We believe that the induction of inflammation in susceptible tumors, possibly coupled with cytotoxic interventions may be an important component in the use of retinoids to treat human squamous cancers. ^

Dynamic Remodeling of the Stressed Heart: Role of Protein Degradation Pathways

The heart is a remarkable organ. In order to maintain its function, it remodels in response to a variety of environmental stresses, including pressure overload, volume overload, mechanical or pharmacological unloading and hormonal or metabolic disturbances. All these responses are linked to the inherent capacity of the heart to rebuild itself. Particularly, cardiac pressure overload activates signaling pathways of both protein synthesis and degradation. While much is known about regulators of protein synthesis, little is known about regulators of protein degradation in hypertrophy. The ubiquitin-proteasome system (UPS) selectively degrades unused and abnormal intracellular proteins. I speculated that the UPS may play an important role in both qualitative and quantitative changes in the composition of heart muscle during hypertrophic remodeling. My study hypothesized that cardiac remodeling in response to hypertrophic stimuli is a dynamic process that requires activation of highly regulated mechanisms of protein degradation as much as it requires protein synthesis. My first aim was to adopt a model of left ventricular hypertrophy and determine its gene expression and structural changes. Male Sprague-Dawley rats were submitted to ascending aortic banding and sacrificed at 7 and 14 days after surgery. Sham operated animals served as controls. Effective aortic banding was confirmed by hemodynamic assessment by Doppler flow measurements in vivo. Banded rats showed a four-fold increase in peak stenotic jet velocities. Histomorphometric analysis revealed a significant increase in myocyte size as well as fibrosis in the banded animals. Transcript analysis showed that banded animals had reverted to the fetal gene program. My second aim was to assess if the UPS is increased and transcriptionally regulated in hypertrophic left ventricular remodeling. Protein extracts from the left ventricles of the banded and control animals were used to perform an in vitro peptidase assay to assess the overall catalytic activity of the UPS. The results showed no difference between hypertrophied and control animals. Transcript analysis revealed decreases in transcript levels of candidate UPS genes in the hypertrophied hearts at 7 days post-banding but not at 14 days. However, protein expression analysis showed no difference at either time point compared to controls. These findings indicate that elements of the UPS are downregulated in the early phase of hypertrophic remodeling and normalizes in a later phase. The results provide evidence in support of a dynamic transcriptional regulation of a major pathway of intracellular protein degradation in the heart. The discrepancy between transcript levels on the one hand and protein levels on the other hand supports post-transcriptional regulation of the UPS pathway in the hypertrophied heart. The exact mechanisms and the functional consequences remain to be elucidated.

Stromal derived growth factor alpha (SDF-1α) induces the differentiation of bone marrow progenitor cells (BMCs) into pericytes by regulating platelet derived growth factor B (PDGF-B) transcription

We previously demonstrated that bone marrow cells (BMCs) migrate to TC71 and A4573 Ewing’s sarcoma tumors where they can differentiate into endothelial cells (ECs) and pericytes and, participate in the tumor vascular development. This process of neo-vascularization, known as vasculogenesis, is essential for Ewing’s sarcoma growth with the soluble vascular endothelial growth factor, VEGF165, being the chemotactic factor for BMC migration to the tumor site. Inhibiting VEGF165 in TC71 tumors (TC/siVEGF7-1) inhibited BMC infiltration to the tumor site and tumor growth. Introducing the stromal-derived growth factor (SDF-1α) into the TC/siVEGF7-1 tumors partially restored vasculogenesis with infiltration of BMCs to a perivascular area where they differentiated into pericytes and rescued tumor growth. RNA collected from the SDF-1α-treated TC/siVEGF7-1 tumors also revealed an increase in platelet-derived growth factor B (PDGF-B) mRNA levels. PDGF-B expression is elevated in several cancer types and the role of PDGF-B and its receptor, PDGFR-β, has been extensively described in the process of pericyte maturation. However, the mechanisms by which PDGF-B expression is up-regulated during vascular remodeling and the process by which BMCs differentiate into pericytes during tumor vasculogenesis remain areas of investigation. In this study, we are the first to demonstrate that SDF-1α regulates the expression of PDGF-B via a transcriptional mechanism which involves binding of the ELK-1 transcription factor to the pdgf-b promoter. We are also first to validate the critical role of the SDF-1α/PDGF-B pathway in the differentiation of BMCs into pericytes both in vitro and in vivo. SDF-1α up-regulated PDGF-B expression in both TC/siVEGF7-1 and HEK293 cells. In contrast, down-regulating SDF-1α, down-regulated PDGF-B. We cloned the 2 kb pdgf-b promoter fragment into the pGL3 reporter vector and showed that SDF-1α induced pdgf-b promoter activity. We used chromatin immunoprecipitation (ChIP) and demonstrated that the ELK-1 transcription factor bound to the pdgf-b promoter in response to SDF-1α stimulation in both TC/siVEGF7-1 and HEK293 cells. We collected BMCs from the hind femurs of mice and cultured the cells in medium containing SDF-1α and PDGF-B and found that PDGFR-β+ BMCs differentiated into NG2 and desmin positive pericytes in vitro. In contrast, inhibiting SDF-1α and PDGF-B abolished this differentiation process. In vivo, we injected TC71 or A4573 tumor-bearing mice with the SDF-1α antagonist, AMD3100 and found that inhibiting SDF-1α signaling in the tumor microenvironment decreased the tumor microvessel density, decreased the tumor blood vessel perfusion and, increased tumor cell apoptosis. We then analyzed the effect of AMD3100 on vasculogenesis of Ewing’s sarcoma and found that BMCs migrated to the tumor site where they differentiated into ECs but, they did not form thick perivascular layers of NG2 and desmin positive pericytes. Finally, we stained the AMD3100-treated tumors for PDGF-B and showed that inhibiting SDF-1α signaling also inhibited PDGF-B expression. All together, these findings demonstrated that the SDF-1α/PDGF-B pathway plays a critical role in the formation of BM-derived pericytes during vasculogenesis of Ewing’s sarcoma tumors.

Cardiolipin biosynthesis and remodeling enzymes are altered during development of heart failure.

Cardiolipin (CL) is responsible for modulation of activities of various enzymes involved in oxidative phosphorylation. Although energy production decreases in heart failure (HF), regulation of cardiolipin during HF development is unknown. Enzymes involved in cardiac cardiolipin synthesis and remodeling were studied in spontaneously hypertensive HF (SHHF) rats, explanted hearts from human HF patients, and nonfailing Sprague Dawley (SD) rats. The biosynthetic enzymes cytidinediphosphatediacylglycerol synthetase (CDS), phosphatidylglycerolphosphate synthase (PGPS) and cardiolipin synthase (CLS) were investigated. Mitochondrial CDS activity and CDS-1 mRNA increased in HF whereas CDS-2 mRNA in SHHF and humans, not in SD rats, decreased. PGPS activity, but not mRNA, increased in SHHF. CLS activity and mRNA decreased in SHHF, but mRNA was not significantly altered in humans. Cardiolipin remodeling enzymes, monolysocardiolipin acyltransferase (MLCL AT) and tafazzin, showed variable changes during HF. MLCL AT activity increased in SHHF. Tafazzin mRNA decreased in SHHF and human HF, but not in SD rats. The gene expression of acyl-CoA: lysocardiolipin acyltransferase-1, an endoplasmic reticulum MLCL AT, remained unaltered in SHHF rats. The results provide mechanisms whereby both cardiolipin biosynthesis and remodeling are altered during HF. Increases in CDS-1, PGPS, and MLCL AT suggest compensatory mechanisms during the development of HF. Human and SD data imply that similar trends may occur in human HF, but not during nonpathological aging, consistent with previous cardiolipin studies.

FUNCTION OF ZNF668 IN CANCER DEVELOPMENT

Human cancer develops as a result of accumulation of mutations in oncogenes and tumor suppressor genes. Zinc finger protein 668 (ZNF668) has recently been identified and validated as one of the highly mutated genes in breast cancer, but its function is entirely unknown. Here, we report two major functions of ZNF668 in cancer development. (1) ZNF668 functions as a tumor suppressor by regulating p53 protein stability and function. We demonstrate that ZNF668 is a nucleolar protein that physically interacts with both MDM2 and p53. By binding to MDM2, ZNF668 regulates MDM2 autoubiquitination and prevents MDM2-mediated p53 ubiquitination and degradation; ZNF668 deficiency impairs DNA damage-induced p53 stabilization. Notably, ZNF668 effectively suppresses breast cancer cell proliferation and transformation in vitro and tumorigenicity in vivo. Consistently, ZNF668 knockdown readily transforms normal mammary epithelial cells. Together, our studies identify ZNF668 as a novel breast tumor suppressor gene that acts at least in part by regulating the stability and function of p53. (2) ZNF668 functions as a DNA repair protein by regulating histone acetylation. DNA repair proteins need to access the chromatin by chromatin modification or remodeling to use DNA template within chromatin. Dynamic posttranslational modifications of histones are critical for cells to relax chromatin in DNA repair. However, the precise underlying mechanism mediating enzymes responsible for these modifications and their recruitment to DNA lesions remains poorly understood. We observed ZNF668 depletion causes impaired chromatin relaxation as a result of impaired DNA-damage induced histone H2AX hyper-acetylation. This results in the decreased recruitment of repair proteins to DNA lesions, defective homologous recombination (HR) repair and impaired cell survival after DNA damage, albeit with the presence of a functional ATM/ATR dependent DNA-damage signaling cascade. Importantly, the impaired loading of repair proteins and the defect in DNA repair in ZNF668-deficient cells can be counteracted by chromatin relaxation, indicating that the DNA-repair defect that was observed in the absence of ZNF668 is due to impeded chromatin accessibility at sites of DNA breaks. Our findings therefore identify ZNF668 as a key molecule that links chromatin relaxation with response to DNA damage in the control of DNA repair.

DEFINITION OF THE LANDSCAPE OF CHROMATIN STRUCTURE AT THE FRATAXIN GENE IN FRIEDREICH’S ATAXIA

Friedreich’s ataxia (FRDA) is caused by the transcriptional silencing of the frataxin (FXN) gene. FRDA patients have expansion of GAA repeats in intron 1 of the FXN gene in both alleles. A number of studies demonstrated that specific histone deacetylase inhibitors (HDACi) affect either histone modifications at the FXN gene or FXN expression in FRDA cells, indicating that the hyperexpanded GAA repeat may facilitate heterochromatin formation. However, the correlation between chromatin structure and transcription at the FXN gene is currently limited due to a lack of more detailed analysis. Therefore, I analyzed the effects of the hyperexpanded GAA repeats on transcription status and chromatin structure using lymphoid cell lines derived from FRDA patients. Using chromatin immunoprecipitation and quantitative PCR, I observed significant changes in the landscape of histone modifications in the vicinity of the GAA tract in FRDA cells relative to control cells. Similar epigenetic changes were observed in GFP reporter construct containing 560 GAA repeats. Further, I detected similar levels of FXN pre-mRNA at a region upstream of hyperexpanded GAA repeats in FRDA and control cells, indicating similar efficiency of transcription initiation in FRDA cells. I also showed that histone modifications associated with hyperexpanded GAA repeats are independent of transcription progression using the GFP reporter system. My data strongly support evidence that FXN deficiency in FRDA patients is consequence of defective transition from initiation to elongation of FXN transcription due to heterochromatin-like structures formed in the proximity of the hyperexpanded GAAs.