CSB Gene Research Articles

The role of RAD51 regulators and variants in primary ovarian insufficiency, endometriosis,and polycystic ovary syndrome.

The study of RAD51 regulators in female reproductive diseases has novel biomarker potential and implications for therapeutic advancement. Regulators of RAD51 play important roles in maintaining genome integrity and variations in these genes have been identified in female reproductive diseases including primary ovarian insufficiency (POI), endometriosis, and polycystic ovary syndrome (PCOS). RAD51 modulators change RAD51 activity in homologous recombination, replication stress, and template switching pathways. However, molecular implications of these proteins in primary ovarian insufficiency, endometriosis, and polycystic ovary syndrome have been understudied. For each reproductive disease, we provide its definition, current diagnostic and therapeutic treatment strategies, and associated genetic variations. Variants were discovered in RAD51, and regulators including DMC1, RAD51B, SWS1, SPIDR, XRCC2and BRCA2 linked with POI. Endometriosis is associated with variants in XRCC3, BRCA1and CSB genes. Variants in BRCA1 were associated with PCOS.Our analysis identifiednovel biomarkers for POI (DMC1 and RAD51B) and PCOS (BRCA1). Further biochemical and cellular analyses of RAD51 regulator functions in reproductive disorders will advance our understanding of the pathogenesis of these diseases.

PHARMACOLOGIC AMPK ACTIVATION IMPROVES HEALTHSPAN AND LIFESPAN IN NORMAL AND PREMATURE AGING MODELS

Abstract Cockayne Syndrome (CS) is a rare autosomal recessive childhood disease, caused by mutations in CSA (ERCC8) or CSB (ERCC6) genes leading to defective DNA damage repair. CS characteristics include UV sensitivity and premature aging. Deficiencies in CSA or CSB may lead to impaired AMP-activated protein kinase (AMPK) activation. Notably, AMPK-activating interventions have shown promise in preclinical CS mouse models. We tested whether MK8722, an AMPK activator, could mitigate CS phenotypes in a C. elegans model. Our preliminary data show that MK-8722 increases lifespan in wild-type and CS worm model. We also tested the effect of MK-8722 on brood size as a fitness parameter and showed that it does not change the total brood size. We also showed that treating aged mice with MK-8722 improves multiple measures of healthspan including body composition, grip strength and age-associated inflammation. These data suggest that AMPK activation may be a promising therapeutic avenue for both normal and CS-associated aging phenotypes

Live cell transcription-coupled nucleotide excision repair dynamics revisited

Transcription–blocking lesions are specifically targeted by transcription-coupled nucleotide excision repair (TC-NER), which prevents DNA damage-induced cellular toxicity and maintains proper transcriptional processes. TC-NER is initiated by the stalling of RNA polymerase II (RNAPII), which triggers the assembly of TC-NER-specific proteins, namely CSB, CSA and UVSSA, which collectively control and drive TC-NER progression. Previous research has revealed molecular functions for these proteins, however, exact mechanisms governing the initiation and regulation of TC-NER, particularly at low UV doses have remained elusive, partly due to technical constraints. In this study, we employ knock-in cell lines designed to target the endogenous CSB gene locus with mClover, a GFP variant. Through live cell imaging, we uncover the intricate molecular dynamics of CSB in response to physiologically relevant UV doses. We showed that the DNA damage-induced association of CSB with chromatin is tightly regulated by the CSA-containing ubiquitin-ligase CRL complex (CRL4CSA). Combining the CSB-mClover knock-in cell line with SILAC-based GFP-mediated complex isolation and mass-spectrometry-based proteomics, revealed novel putative CSB interactors as well as discernible variations in complex composition during distinct stages of TC-NER progression. Our work not only provides molecular insight into TC-NER, but also illustrates the versatility of endogenously tagging fluorescent and affinity tags.

Nucleotide excision repair in Human cell lines lacking both XPC and CSB proteins.

Nucleotide excision repair removes UV-induced DNA damage through two distinct sub-pathways, global repair and transcription-coupled repair (TCR). Numerous studies have shown that in human and other mammalian cell lines that the XPC protein is required for repair of DNA damage from nontranscribed DNA via global repair and the CSB protein is required for repair of lesions from transcribed DNA via TCR. Therefore, it is generally assumed that abrogating both sub-pathways with an XPC-/-/CSB-/- double mutant would eliminate all nucleotide excision repair. Here we describe the construction of three different XPC-/-/CSB-/- human cell lines that, contrary to expectations, perform TCR. The XPC and CSB genes were mutated in cell lines derived from Xeroderma Pigmentosum patients as well as from normal human fibroblasts and repair was analyzed at the whole genome level using the very sensitive XR-seq method. As predicted, XPC-/- cells exhibited only TCR and CSB-/- cells exhibited only global repair. However, the XPC-/-/CSB-/- double mutant cell lines, although having greatly reduced repair, exhibited TCR. Mutating the CSA gene to generate a triple mutant XPC-/-/CSB-/-/CSA-/- cell line eliminated all residual TCR activity. Together, these findings provide new insights into the mechanistic features of mammalian nucleotide excision repair.

ATM DNA METHYLATION ASSOCIATED WITH POOR PROGNOSIS AND LEUKEMIA TRANSFORMATION IN MYELODYSPLASTIC SYNDROME PATIENTS

Myelodysplastic syndrome (MDS) is a clonal disease of hematopoietic progenitors with a risk of progressing to acute myeloid leukemia. MDS etiology is mainly related to age, but one of the risk factors is exposure to pesticides that causes aberrant reprogramming of the genome by DNA methylation. The aim of this work was evaluating the methylation profile of genes acting in the single-strand DNA (ssDNA) repair (CSA, CSB, XPA, XPC and XPG) and double-strand DNA (dsDNA) repair (ATM, BRCA1, BRCA2, LIG4 and RAD51), and its relationship with gene expression. The analysis was performed using bone marrow samples from 56 MDS patients, 69 farmers exposed to pesticides and 10 controls without exposition or MDS. The methylation profile and gene expression were evaluated using pyro-sequencing technique and quantitative real-time PCR. Regarding ssDNA repair genes, in cases with MDS, patients under 60 years old showed a higher methylation for the XPA and CSB genes than patients over 60 years old (p = 0.008; p = 0.017). Furthermore, XPA showed increased methylation levels in initial forms of MDS (p = 0.019). The MDS group presented a higher percentage of XPA methylation than exposed farmers (p = 0.000) and the control group (p = 0.011). In the prognostic classification, lower risk presented higher methylation than high-risk (p = 0.027). Among the dsDNA repair genes methylation in MDS patients, only ATM gene showed significant results with increase of methylation levels in (1) males (p = 0.037); (2) patients aged up to 60 years (p = 0.002) and (3) those who progressed to AML (p = 0.016). Regarding the methylation between the groups of exposed farmers, MDS advanced forms and controls, we also detected significant differences with levels always increased in the control group: ATM (control group vs. farmers p = 0.000), BRCA1 exposed vs. control group, p = 0.001; MDS vs. control, p = 0.001;), BRCA2 (farmers vs. control, p = 0.027; MDS vs. control, p = 0.004), LIG4 (farmers vs. control, p = 0.001; MDS vs. control, p = 0.012) and RAD51 (farmers vs. controls, p = 0.047). The correlation analysis between methylation and expression showed a positive and strong correlation for LIG4 gene in the advanced forms MDS group (r = 0.798) and a positive and weak correlation for BRCA1 gene in the exposed farmers group (r = 0.340). Surprisingly, in the ssDNA repair genes, the expression and methylation of both farmers and MDS patients were not correlated, suggesting that the change in the expression of these genes is not motivated by the DNA methylation mechanism. Furthermore, the analysis of dsDNA repair genes in this study corroborates the phenomenon of relative hypermethylation in some DNA repair genes in MDS.

A matter of delicate balance: Loss and gain of Cockayne syndrome proteins in premature aging and cancer.

DNA repair genes are critical for preserving genomic stability and it is well established that mutations in DNA repair genes give rise to progeroid diseases due to perturbations in different DNA metabolic activities. Cockayne Syndrome (CS) is an autosomal recessive inheritance caused by inactivating mutations in CSA and CSB genes. This review will primarily focus on the two Cockayne Syndrome proteins, CSA and CSB, primarily known to be involved in Transcription Coupled Repair (TCR). Curiously, dysregulated expression of CS proteins has been shown to exhibit differential health outcomes: lack of CS proteins due to gene mutations invariably leads to complex premature aging phenotypes, while excess of CS proteins is associated with carcinogenesis. Thus it appears that CS genes act as a double-edged sword whose loss or gain of expression leads to premature aging and cancer. Future mechanistic studies on cell and animal models of CS can lead to potential biological targets for interventions in both aging and cancer development processes. Some of these exciting possibilities will be discussed in this review in light of the current literature.

Effects of Oxygen Tension for Membrane Lipidome Remodeling of Cockayne Syndrome Cell Models.

Oxygen is important for lipid metabolism, being involved in both enzymatic transformations and oxidative reactivity, and is particularly influent when genetic diseases impair the repair machinery of the cells, such as described for Cockayne syndrome (CS). We used two cellular models of transformed fibroblasts defective for CSA and CSB genes and their normal counterparts, grown for 24 h under various oxygen tensions (hyperoxic 21%, physioxic 5% and hypoxic 1%) to examine the fatty acid-based membrane remodeling by GC analysis of fatty acid methyl esters derived from membrane phospholipids. Overall, we first distinguished differences due to oxygen tensions: (a) hyperoxia induced a general boost of desaturase enzymatic activity in both normal and defective CSA and CSB cell lines, increasing monounsaturated fatty acids (MUFA), whereas polyunsaturated fatty acids (PUFA) did not undergo oxidative consumption; (b) hypoxia slowed down desaturase activities, mostly in CSA cell lines and defective CSB, causing saturated fatty acids (SFA) to increase, whereas PUFA levels diminished, suggesting their involvement in hypoxia-related signaling. CSB-deprived cells are the most sensitive to oxidation and CSA-deprived cells are the most sensitive to the radical-based formation of trans fatty acids (TFA). The results point to the need to finely differentiate biological targets connected to genetic impairments and, consequently, suggest the better definition of cell protection and treatments through accurate molecular profiling that includes membrane lipidomes.

Immunofluorescence studies to dissect the impact of Cockayne syndrome A alterations on the protein interaction and cellular localization

BackgroundCockayne syndrome (CS), which was discovered by Alfred Cockayne nearly 75 years ago, is a rare autosomal recessive disorder characterized by growth failure, neurological dysfunction, premature aging, and other clinical features including microcephaly, ophthalmologic abnormalities, dental caries, and cutaneous photosensitivity. These alterations are caused by mutations in the CSA or CSB genes, both of which are involved in transcription-coupled nucleotide excision repair (TC-NER), the sub-pathway of NER that rapidly removes UV-induced DNA lesions which block the progression of the transcription machinery in the transcribed strand of active genes. Several studies assumed that CSA and CSB genes can play additional roles outside TC-NER, due to the wide variations in type and severity of the CS phenotype and the lack of a clear relationship between genotype and phenotype. To address this issue, our lab generated isogenic cell lines expressing wild type as well as different versions of mutated CSA proteins, fused at the C-terminus with the Flag and HA epitope tags (CSAFlag-HA). In unpublished data, the identity of the CSA-interacting proteins was determined by mass spectrometry. Among which three subunits (namely, CCT3, CCT8, and TCP1) of the TRiC/CCT complex appeared as novel interactors. TRiC is a chaperonin involved in the folding of newly synthesized or unfolded proteins. The aim of this study is directed to investigate by immunofluorescence analysis the impact of the selected CSA mutations on the subcellular localization of the CSA protein itself as well as on its novel interactors CCT3, CCT8, and TCP1. ResultsWe showed that specific CSA mutations impair the proper cellular localization of the protein, but have no impact on the cellular distribution of the TRiC subunits or CSA/TRiC co-localization. ConclusionWe suggested that the activity of the TRiC complex does not rely on the functionality of CSA.

Oxygen-Dependent Accumulation of Purine DNA Lesions in Cockayne Syndrome Cells.

Cockayne Syndrome (CS) is an autosomal recessive neurodegenerative premature aging disorder associated with defects in nucleotide excision repair (NER). Cells from CS patients, with mutations in CSA or CSB genes, present elevated levels of reactive oxygen species (ROS) and are defective in the repair of a variety of oxidatively generated DNA lesions. In this study, six purine lesions were ascertained in wild type (wt) CSA, defective CSA, wtCSB and defective CSB-transformed fibroblasts under different oxygen tensions (hyperoxic 21%, physioxic 5% and hypoxic 1%). In particular, the four 5′,8-cyclopurine (cPu) and the two 8-oxo-purine (8-oxo-Pu) lesions were accurately quantified by LC-MS/MS analysis using isotopomeric internal standards after an enzymatic digestion procedure. cPu levels were found comparable to 8-oxo-Pu in all cases (3–6 lesions/106 nucleotides), slightly increasing on going from hyperoxia to physioxia to hypoxia. Moreover, higher levels of four cPu were observed under hypoxia in both CSA and CSB-defective cells as compared to normal counterparts, along with a significant enhancement of 8-oxo-Pu. These findings revealed that exposure to different oxygen tensions induced oxidative DNA damage in CS cells, repairable by NER or base excision repair (BER) pathways. In NER-defective CS patients, these results support the hypothesis that the clinical neurological features might be connected to the accumulation of cPu. Moreover, the elimination of dysfunctional mitochondria in CS cells is associated with a reduction in the oxidative DNA damage.

Transcription-coupled nucleotide excision repair is coordinated by ubiquitin and SUMO in response to ultraviolet irradiation.

Cockayne Syndrome (CS) is a severe neurodegenerative and premature aging autosomal-recessive disease, caused by inherited defects in the CSA and CSB genes, leading to defects in transcription-coupled nucleotide excision repair (TC-NER) and consequently hypersensitivity to ultraviolet (UV) irradiation. TC-NER is initiated by lesion-stalled RNA polymerase II, which stabilizes the interaction with the SNF2/SWI2 ATPase CSB to facilitate recruitment of the CSA E3 Cullin ubiquitin ligase complex. However, the precise biochemical connections between CSA and CSB are unknown. The small ubiquitin-like modifier SUMO is important in the DNA damage response. We found that CSB, among an extensive set of other target proteins, is the most dynamically SUMOylated substrate in response to UV irradiation. Inhibiting SUMOylation reduced the accumulation of CSB at local sites of UV irradiation and reduced recovery of RNA synthesis. Interestingly, CSA is required for the efficient clearance of SUMOylated CSB. However, subsequent proteomic analysis of CSA-dependent ubiquitinated substrates revealed that CSA does not ubiquitinate CSB in a UV-dependent manner. Surprisingly, we found that CSA is required for the ubiquitination of the largest subunit of RNA polymerase II, RPB1. Combined, our results indicate that the CSA, CSB, RNA polymerase II triad is coordinated by ubiquitin and SUMO in response to UV irradiation. Furthermore, our work provides a resource of SUMO targets regulated in response to UV or ionizing radiation.

Multisystem analyses of two Cockayne syndrome associated proteins CSA and CSB reveal shared and unique functions

Mutations in the CSA and CSB genes are causative of Cockayne syndrome neurological disorder. Since the identification of indispensable functions of these two proteins in transcription-coupled repair and restoring RNA synthesis following DNA damage, the paradoxical less severe clinical symptoms reported in some CS-A patients have been puzzling. In this study we compared the effects of a CSA or a CSB defect at the levels of the cell and the intact organism. We showed that CSA-deficient zebrafish embryos exhibited modest hypersensitive to UV damage than CSB depletion. We found that loss of CSA can effectively release aggregation of mutant crystallin proteins in vitro. We described the opposite effect of CSA and CSB on neuritogenesis and elucidated the differentiated gene expression pathways regulated by these two proteins. Our data demonstrate convergent and divergent roles for CSA and CSB in DNA repair and transcription regulation and provide potential explanations for the observed differences between CS-A and CS-B patients.

Cockayne syndrome group B deficiency reduces H3K9me3 chromatin remodeler SETDB1 and exacerbates cellular aging.

Cockayne syndrome is an accelerated aging disorder, caused by mutations in the CSA or CSB genes. In CSB-deficient cells, poly (ADP ribose) polymerase (PARP) is persistently activated by unrepaired DNA damage and consumes and depletes cellular nicotinamide adenine dinucleotide, which leads to mitochondrial dysfunction. Here, the distribution of poly (ADP ribose) (PAR) was determined in CSB-deficient cells using ADPr-ChAP (ADP ribose-chromatin affinity purification), and the results show striking enrichment of PAR at transcription start sites, depletion of heterochromatin and downregulation of H3K9me3-specific methyltransferases SUV39H1 and SETDB1. Induced-expression of SETDB1 in CSB-deficient cells downregulated PAR and normalized mitochondrial function. The results suggest that defects in CSB are strongly associated with loss of heterochromatin, downregulation of SETDB1, increased PAR in highly-transcribed regions, and mitochondrial dysfunction.

Cockayne Syndrome

Cockayne syndrome (CS) is an autosomal recessive disorder characterized by severe photosensitive genodermatosis that is associated with premature aging caused by defects in the UV-induced DNA damage repair system, particularly the transcription-coupled nucleotide excision repair. The clinical features of CS include photosensitivity, a characteristic senile face, significant developmental abnormalities, such as short stature, underweight, and microcephaly, progressive cachexia, severe visual impairment, and sensorineural deafness. CS is clinically classified into type I (classical type), type II (congenital or severe type) and type III (late-onset or adult-onset type). Additionally, there exists a rare form of xerodema pigmentosum-Cockayne syndrome (XP/CS) complex. The incidence of CS is 2.7 in 1,000,000 individuals in Japan and 90% of the cases are type I. Unlike XP, in CS, skin cancer is not known to occur in areas of skin exposed to sunlight. However, we observed a case where solar keratosis developed in adult-onset CS patients (CS type III) with a pathological mutation in the CSB gene. In XP/CS, patients easily develop skin cancer from early childhood in areas of the skin exposed to sunlight.

Influence of chromatin remodeling in the removal of UVC-induced damage in TCR proficient and deficient Chinese hamster cells

In mammalian cells, nucleotide excision repair system is constituted of two sub-pathways, global genomic repair (GGR) and transcription coupled repair (TCR). Deficiency of TCR pathway leads to Cockyane syndrome (CS) which is a rare human autosomal recessive disorder. Owing to the pivotal role of CSB gene in TCR, it’s mutation causes severe repair and transcriptional defects in CSB patients. CSB protein belongs to the ATP chromatin remodeling complex, hence presumably an improper chromatin remodeling in CSB cells could be at the source of inefficient removal of pyrimidine dimers (CPDs) after UVC exposure in these patients. In this study, we evaluated the role of chromatin remodeling process on UVC induced CPDs and the ensuing effect on chromosomal aberrations in UV61 cells (TCR deficient) and its parental cell line, AA8 (TCR proficient). We observed that post 2 h UVC irradiation, both cell lines underwent pronounced chromatin relaxation but was lower in CSB deficient UV61 cells. Since the deficiency in chromatin remodeling in CSB-mutated cells was accompanied by a decrease in the histone acetylation level, the histone deacetylase inhibitor trichostatin A (TSA) was employed to improve the removal of UVC-induced lesions by increasing the histone acetylation level. Contrary to expectations, TSA increased the induction of chromosomal aberrations and apoptotic cells along with amounts of CPDs after UVC-irradiation, indicating that changes in histone acetylation levels might contribute to the failure in the removal of UVC-induced lesions. Also, it has been shown earlier that the expression of genes regulated by CSB is affected by the increase in the acetylation level produced by TSA. Taken all together, we hypothesize that failure in the removal of UVC induced lesions in CSB-deficient cells can be caused by an imbalance in histone acetylation levels leading to chromatin conformation changes and hence interaction defects among repair proteins and DNA lesions.

International Investigative Dermatology 2018 Meeting: Promoting Global Skin Biology and Skin Disease Research

International Investigative Dermatology 2018 Meeting: Promoting Global Skin Biology and Skin Disease Research

741 UV stress directs Cockayne Syndrome (CS) B protein to the centrosome to control α-tubulin acetylation and autophagy

The hereditary progeria CS is predominantly caused by mutations in the csb gene. CS has originally been classified as a DNA repair disorder, but we have previously shown that also autophagy is dysfunctional in CS and that this autophagic block may be overcome by treatment with the pan HDAC inhibitor SAHA. Importantly, SAHA, which is a registered drug, was capable of rescuing the skin phenotype of UV-irradiated CSB-deficient mice. In the present study we therefore asked how SAHA treatment can improve autophagy in CS. One essential step in autophagy is acetylation of α-tubulin which is promotes effective fusion of autophagosomes with lysosomes. We now report that in CS models from three different species autophagic dysfunction was associated with decreased acetyl-α-tubulin levels, and that SAHA treatment improved both α-tubulin acetylation and autophagy. Further analysis in human fibroblasts showed that upon exposure to UV stress, wildtype, but also mutant CSB protein was recruited to the centrosome where it co-localized with dynein and HDAC6. This indicates that lack of α-tubulin acetylation in CS is not due to deficient CSB translocation, but most likely results from the disturbed interaction of CSB with HDAC6, dynein and/or yet to be defined centrosomal proteins. Accordingly, inhibition of HDAC6 was sufficient to enhance autophagic flux in CSB-deficient fibroblasts, and this effect critically depended on tubulin integrity and dynein function. Our results suggest that (i) the CSB protein has an important function outside of the nucleus, where it regulates autophagy by interacting with centrosomal proteins to acetylate α-tubulin and that (ii) SAHA treatment works in CS by overcoming deficient α-tubulin acetylation.

Cockayne syndrome group A and B proteins converge on transcription-linked resolution of non-B DNA

Cockayne syndrome is a neurodegenerative accelerated aging disorder caused by mutations in the CSA or CSB genes. Although the pathogenesis of Cockayne syndrome has remained elusive, recent work implicates mitochondrial dysfunction in the disease progression. Here, we present evidence that loss of CSA or CSB in a neuroblastoma cell line converges on mitochondrial dysfunction caused by defects in ribosomal DNA transcription and activation of the DNA damage sensor poly-ADP ribose polymerase 1 (PARP1). Indeed, inhibition of ribosomal DNA transcription leads to mitochondrial dysfunction in a number of cell lines. Furthermore, machine-learning algorithms predict that diseases with defects in ribosomal DNA (rDNA) transcription have mitochondrial dysfunction, and, accordingly, this is found when factors involved in rDNA transcription are knocked down. Mechanistically, loss of CSA or CSB leads to polymerase stalling at non-B DNA in a neuroblastoma cell line, in particular at G-quadruplex structures, and recombinant CSB can melt G-quadruplex structures. Indeed, stabilization of G-quadruplex structures activates PARP1 and leads to accelerated aging in Caenorhabditis elegans In conclusion, this work supports a role for impaired ribosomal DNA transcription in Cockayne syndrome and suggests that transcription-coupled resolution of secondary structures may be a mechanism to repress spurious activation of a DNA damage response.

565 Aberrant histone acetylation is linked to lysosomal and autophagic dysfunction in Cockayne syndrome (CS)

Cockayne syndrome (CS) is a progeroid disease mainly caused by mutations in the CSB gene. As CS patients show UV hypersensitivity and cells lacking functional CSB protein are defective in transcription coupled DNA repair, CS is usually regarded as a DNA repair deficiency syndrome. Importantly, clinical hallmarks of CS suggest additional biological functions of CSB apart from DNA repair. In keeping with this assumption we have previously provided evidence for disturbed autophagy in CSB and that treatment of CSB deficient (CSBdef) mice with the HDAC inhibitor SAHA rescued their skin phenotype including inflammation, fibrosis and loss of subcutaneous fat. In the present study we link these two findings. We show that the development of the skin phenotype in CSBdef mice was associated with a blockade in autophagy due to lysosomal dysfunction as revealed by accumulation of autophagy- and lysosome-related proteins in their degenerated subcutis. Similar to mice, autophagic/lysosomal blockade was also present in CSBdef primary human fibroblasts and C. elegans. Moreover, CSBdef fibroblasts displayed reduced acetylation of H3K56 and H4K16, two histone lysine residues directly linked to regulation of autophagy. In line with that, we also detected high levels of HDAC1 and HDAC3 proteins in these cells and murine skin which are not only capable of deacetylating H3K56 and H4K16, respectively, but are also involved in regulation of autophagy suggesting involvement of an imbalanced acetylation in autophagic dysfunction and skin pathology. Accordingly, application of SAHA increased acetylation of both lysine residues in CSBdef fibroblasts and improved autophagic/lysosomal function in these cells, C. elegans and CSBdef mice. These results indicate a possible mechanistic link between epigenetic alterations, autophagy dysfunction and loss of subcutaneous fat in CS.

Expression of Placental Members of the Human Growth Hormone Gene Family Is Increased in Response to Sequential Inhibition of DNA Methylation and Histone Deacetylation.

The genes coding for human (h) chorionic somatomammotropin (CS), hCS-A and hCS-B, and placental growth hormone (GH-V), hGH-V, are located at a single locus on chromosome 17. Efficient expression of these placental genes has been linked to local regulatory (5′ P and 3′ enhancer) sequences and a remote locus control region (LCR), in part, through gene transfer in placental and nonplacental tumor cells. However, low levels of endogenous hCS/GH-V transcripts are reported in the same cells compared with term placenta, suggesting that chromatin structure, or regulatory region accessibility, versus transcription factor availability contributes to the relatively low levels. To assess individual hCS-A, CS-B, and GH-V gene expression in placental and nonplacental tumor cells and the effect of increasing chromatin accessibility by inhibiting DNA methylation and histone deacetylation using 5-aza-2′-deoxycytidine (azadC) and trichostatin A (TSA). Low levels of hCS-A, CS-B, and GH-V were detected in placental and nonplacental tumor cells compared with term placenta. A significant >5-fold increase in activity was seen in placental, but not nonplacental, cells transfected with hybrid hCS promoter luciferase genes containing 3′ enhancer sequences. Pretreatment of placental JEG-3 cells with azadC resulted in a >10-fold increase in hCS-A, CS-B, and GH-V RNA levels with TSA treatment compared with TSA treatment alone. This effect was specific as reversing the treatment regimen did not have the same effect. An assessment of hyperacetylated H3/H4 in JEG-3 cells treated with azadC and TSA versus TSA alone revealed significant increases consistent with a more open chromatin structure, including the hCS 3′ enhancer sequences and LCR. These observations suggest that accessibility of remote and local regulatory regions required for efficient placental hGH/CS expression can be restricted by DNA methylation and histone acetylation status. This includes restricting access of the hCS 3′ enhancer sequences to available placental enhancer transcription factors.

601. Gene Therapy for Cockayne Syndrome

Cockayne Syndrome (CS) is a rare autosomal recessive genetic disorder. The majority of CS cases involve mutations in either the CSA (25%) or CSB (75%) gene, leading to defective transcription-coupled nucleotide excision repair and RNA polymerase II mediated transcription. Clinically this presents as progressive degeneration of the central nervous system, retina, cardiovascular system, and cochlea, causing mental retardation, post-natal growth defects, pigmented retinopathy, cataracts, dermal UV sensitivity, organ dysfunction and shortened life expectancy.CSA and CSB single mutant mice display retinal degeneration with age and ocular sensitivity to ionizing radiation. Because limited publications on these mouse models exist we began by characterizing the retinal dysfunction through histology and electroretinography (ERG). We found that the retinal degeneration in CSA and CSB mutant mice (C57Bl6 background) was occurring over a slower time course (over 1 year) than previously reported. Therefore we aimed to increase the rate of retinal degeneration in these animals using UV light exposure, whole body radiation, or high intensity light exposure. Our goal was to increase the speed of retinal degeneration to a timeframe that is more readily studied for the purposes of gene therapy correction. Preliminary data indicates that we can cause a decrease in the scotopic ERG (b-wave) 2 weeks after high-dose ultraviolet light exposure (100000J/m^2), a change not seen in C57Bl6 mice at the same UV dose. There was also a decrease in b-wave amplitude following radiation exposure (2-3 gray) in the CSB mouse model that was not seen in C57Bl6 mice. Histology on these mice is currently pending. Improving the rate of photoreceptor loss will allow us to perform rAAV based gene therapy to deliver the CSB gene back to the retina and ascertain whether UV and radiation resistance can be restored.While single mutant CSA and CSB mice do not have significant neurological deficits, when crossed with XPA deficient mice they develop clinical signs similar to severely affected human patients. Signs in these mice include neurologic deficits, poor weight gain and death around postnatal day 21. These mice could be ideal candidates to show that correction of both the central nervous system disease as well as the retinal degeneration is possible in a single model. To determine if both the brain and retina could be targeted in the same animal (and therefore model what may be necessary in the human patients) we have looked at targeting both organs simultaneously using either a single IV injection in neonatal mice or a combined intra-cerebral ventricular (ICV) injection and IV injection in the same animal. We found that delivery of the rAAV vector (regardless of serotype tested) into the retro-orbital sinus at postnatal day 1-3 leads to widespread transduction of the retina. Temporal vein delivery postnatal day 1-3 only resulted in transduction of the inner layer of the retina, not the photoreceptors. When ICV and IV injections were combined in neonates widespread brain and retinal transduction was achieved.These preliminary data indicate that a murine model for retinal gene therapy of CS is feasible and that potentially both the retina and brain could be targeted in a single model.