Wolf-Hirschhorn Syndrome Candidate Research Articles

Wolf-Hirschhorn syndrome candidate 1-like 1 epigenetically regulates nephrin gene expression.

Altered expression of nephrin underlies the pathophysiology of proteinuria in both congenital and acquired nephrotic syndrome. However, the epigenetic mechanisms of nephrin gene regulation remain elusive. Here, we show that Wolf-Hirschhorn syndrome candidate 1-like 1 long form (WHSC1L1-L) is a novel epigenetic modifier of nephrin gene regulation. WHSC1L1-L was associated with histone H3K4 and H3K36 in human embryonic kidney cells. WHSC1L1-L gene was expressed in the podocytes, and functional protein product was detected in these cells. WHSC1L1-L was found to bind nephrin but not other podocyte-specific gene promoters, leading to its inhibition/suppression, abrogating the stimulatory effect of WT1 and NF-κB. Gene knockdown of WHSC1L1-L in primary cultured podocytes accelerated the transcription of nephrin but not CD2AP. An in vivo zebrafish study involving the injection of Whsc1l1 mRNA into embryos demonstrated an apparent reduction of nephrin mRNA but not podocin and CD2AP mRNA. Immunohistochemistry showed that both WHSC1L1-L and nephrin emerged at the S-shaped body stage in glomeruli. Immunofluorescence and confocal microscopy displayed WHSC1L1 to colocalize with trimethylated H3K4 in the glomerular podocytes. Chromatin immunoprecipitation assay revealed the reduction of the association of trimethylated H3K4 at the nephrin promoter regions. Finally, nephrin mRNA was upregulated in the glomerulus at the early proteinuric stage of mouse nephrosis, which was associated with the reduction of WHSC1L1. In conclusion, our results demonstrate that WHSC1L1-L acts as a histone methyltransferase in podocytes and regulates nephrin gene expression, which may in turn contribute to the integrity of the slit diaphragm of the glomerular filtration barrier.

Epigenetic activation of WHSC1 functions as an oncogene and is associated with poor prognosis in cervical cancer.

Overexpression of Wolf-Hirschhorn syndrome candidate1 (WHSC1) is commonly observed in various types of tumors. However, the potential mechanism responsible for this molecular event is poorly understood. In the present study, we found that the mRNA levels of WHSC1 were significantly increased in cervical cancer cells, and that CpG sites were almost fully methylated in HaCaT cells, but partially methylated in HeLa and C33A cells. Clinically, the results of quantitative methylation-specific PCR (QMSP) and quantitative real-time PCR (qRT-PCR), showed that methylation levels of the WHSC1 gene were significantly decreased in cervical cancer tumors and inversely correlated with its mRNA expression levels. Both decreased methylation of WHSC1 and increased mRNA were associated with cancer progression and poor prognosis. In addition, overexpression of WHSC1 contributed to cell proliferation, migration and invasion, while cells with WHSC1 knockdown exhibited the opposite effects. AKT/metalloproteinase-2(MMP-2) signaling was activated and inactivated upon overexpression and silencing of WHSC1, respectively. Silencing of WHSC1 also suppressed tumor growth in a xenograft model. In conclusion, WHSC1 is hypomethylated in cervical cancer, and consequent overexpression of WHSC1 mRNA may promote cervical carcinogenesis by activating the AKT/MMP-2 signaling pathway.

Sabotaging of the oxidative stress response by an oncogenic noncoding RNA.

Overexpression of the multiple myeloma set domain (MMSET) Wolf-Hirschhorn syndrome candidate 1 gene, which contains an orphan box H/ACA class small nucleolar RNA, ACA11, in an intron, is associated with several cancer types, including multiple myeloma (MM). ACA11 and MMSET are overexpressed cotranscriptionally as a result of the t(4;14) chromosomal translocation in a subset of patients with MM. RNA sequencing of CD138+ tumor cells from t(4;14)-positive and -negative MM patient bone marrow samples revealed an enhanced oxidative phosphorylation mRNA signature. Supporting these data, ACA11 overexpression in a t(4;14)-negative MM cell line, MM1.S, demonstrated enhanced reactive oxygen species (ROS) levels. In addition, an enhancement of cell proliferation, increased soft agar colony size, and elevated ERK1/2 phosphorylation were observed. This ACA11-driven hyperproliferative phenotype depended on increased ROS levels as exogenously added antioxidants attenuate the increased proliferation. A major transcriptional regulator of the cellular antioxidant response, nuclear factor (erythroid-derived 2)-like 2 (NRF2), shuttled to the nucleus, as expected, in response to ACA11-driven increases in ROS; however, transcriptional up-regulation of some of NRF2's antioxidant target genes was abrogated in the presence of ACA11 overexpression. These data show for the first time that ACA11 promotes proliferation through inhibition of NRF2 function resulting in sustained ROS levels driving cancer cell proliferation.-Mahajan, N., Wu, H.-J., Bennett, R. L., Troche, C., Licht, J. D., Weber, J. D., Maggi, L. B., Jr., Tomasson, M. H. Sabotaging of the oxidative stress response by an oncogenic noncoding RNA.

Wolf-Hirschhorn Syndrome (WHS), A Case Report and Review of Literature Submit Manuscript

Background: Wolf-Hirschhorn Syndrome (WHS) is a congenital malformation syndrome characterized by growth deficiency and varying developmental delays based on genomic deletions and characteristic facies. The majority of WHS cases are caused by a deletion of 4p16.3 regions on chromosome 4, which includes the Wolf-Hirschhorn Syndrome Candidate genes (WHSC1 and WHSC2). WHSC1 protein is required to inhibit DNA damage by regulating the methylation of histones. The diagnosis of WHS is established by detection of a heterozygous deletion of the Wolf-Hirschhorn Syndrome Critical Region (WHSCR) with 4p16.3 at approximately 1.4-1.9 kb from the terminus. The WHSCR region is restricted to a 165-kb interval in the 4p16.3. Some of the associated structural defects, such as cleft lip and palate, occur more frequently in individuals with greater than 3 megabase (Mb) deletions. The quantity of base deletions is an important factor in explaining phenotypic variability WHS. The deletion size has a partial correlation to severity, but not a direct correlation, as the phenotypic affect may be more or less severely affected than would be expected by the size of the deleted megabase. Most individuals affected by this disorder have distinctive facial features, such as a broad, flat nasal bridge and a high forehead described as the “Greek warrior helmet” appearance in which the eyes are protruding and widely spaced. Other facial features include a shortened distance between the nose and the upper lip (a short philtrum), a down-turned mouth, a small chin (micrognathia), poorly formed ears with small holes (pits) of flaps of skin (tags), a small head (microcephaly). Intellectual disability ranges from mild to severe in people with WHS. Compared to people with other forms of intellectual disability, their socialization skills are strong, while verbal, communication, and language skills tend to be weaker. Case: We present the identification during routine obstetric sonographic imaging of growth restricted fetus affected by WHS with co-morbid oligohydramnios and abnormal prenatal obstetrics ultrasound studies, which showed micrognathia, echogenic kidneys and echogenic bowel, in addition to a three-week discrepancy between the estimated ultrasound age and the gestational age by last menstrual period (LMP), compatible with intrauterine growth restriction (IUGR). Prenatal genetic testing demonstrated female karyotype with a satellite chromosome on chromosome 4 short arm and mosaicism for an additional marker chromosome (47, XX, 4ps, +mar (8)/46, XX, 4ps). The prenatal diagnostic approach included fetal sonographic assessment and molecular cytogenetic investigation. After engaging in a multidisciplinary informed consent process at our institution, the patient chose to continue prenatal care and not electively terminate the pregnancy. Conclusion: A 4p deletion on chromosome 4 can be associated with subtle chromosome imbalances in other chromosomes. For example, the t(4;8)(p16;p23) translocation may be undetected in routine cytogenetics, suggesting that this translocation may be more the most frequent translocation after the most common reciprocal translocation in humans, t(11q;22q). The extent of the Mb deleted within the 4p region located on chromosome 4 correlates with the severity of the WHS phenotype. Genetic counseling provides families with information on the nature and implications of inherited genetic disorders, as a means of facilitating shared health care decisions.

Identification of miR-2400 gene as a novel regulator in skeletal muscle satellite cells proliferation by targeting MYOG gene

MicroRNAs play critical roles in skeletal muscle development as well as in regulation of muscle cell proliferation and differentiation. Previous study in our laboratory showed that the expression level of miR-2400, a novel and unique miRNA from bovine, had significantly changed in skeletal muscle-derived satellite cells (MDSCs) during differentiation, however, the function and expression pattern for miR-2400 in MDSCs has not been fully understood. In this report, we firstly identified that the expression levels of miR-2400 were down-regulated during MDSCs differentiation by stem-loop RT-PCR. Over-expression and inhibition studies demonstrated that miR-2400 promoted MDSCs proliferation by EdU (5-ethynyl-2′ deoxyuridine) incorporation assay and immunofluorescence staining of Proliferating cell nuclear antigen (PCNA). Luciferase reporter assays showed that miR-2400 directly targeted the 3′ untranslated regions (UTRs) of myogenin (MYOG) mRNA. These data suggested that miR-2400 could promote MDSCs proliferation through targeting MYOG. Furthermore, we found that miR-2400, which was located within the eighth intron of the Wolf-Hirschhorn syndrome candidate 1-like 1 (WHSC1L1) gene, was down-regulated in MDSCs in a direct correlation with the WHSC1L1 transcript by Clustered regularly interspaced palindromic repeats interference (CRISPRi). In addition, these observations not only provided supporting evidence for the codependent expression of intronic miRNAs and their host genes in vitro, but also gave insight into the role of miR-2400 in MDSCs proliferation.

Retinoic acid inhibits histone methyltransferase Whsc1 during palatogenesis

Cleft lip with or without palate (CL/P) is a common congenital anomaly in humans and is thought to be caused by genetic and environmental factors. However, the epigenetic mechanisms underlying orofacial clefts are not fully understood. Here, we investigate how the overdose of retinoic acid (RA), which can induce cleft palate in mice and humans, regulates histone methyltransferase, Wolf–Hirschhorn syndrome candidate 1 (WHSC1) during palatal development in mice. We treated mouse embryonic fibroblasts (MEFs) with 1 μM all-trans RA and discovered that the global level of H3K36me3 was downregulated and that expression of the H3K36 methyltransferase gene, Whsc1, was reduced. The expression level of WHSC1 in embryonic palatal shelves was reduced during palatogenesis, following maternal administration of 100 mg/kg body weight of RA by gastric intubation. Furthermore, the expression of WHSC1 in palatal shelves was observed in epithelial and mesenchymal cells at all stages, suggesting an important role for palatal development. Our results suggest that the pathogenesis of cleft palate observed after excessive RA exposure is likely to be associated with a reduction in the histone methyltransferase, WHSC1.

WHSC1 promotes oncogenesis through regulation of NIMA-related kinase-7 in squamous cell carcinoma of the head and neck.

Squamous cell carcinoma of the head and neck (SCCHN) is a relatively common malignancy with suboptimal long-term prognosis, thus new treatment strategies are urgently needed. Over the last decade, histone methyltransferases (HMT) have been recognized as promising targets for cancer therapy, but their mechanism of action in most solid tumors, including SCCHN, remains to be elucidated. This study investigated the role of Wolf-Hirschhorn syndrome candidate 1 (WHSC1), an NSD family HMT, in SCCHN. Immunohistochemical analysis of locoregionally advanced SCCHN, dysplastic, and normal epithelial tissue specimens revealed that WHSC1 expression and dimethylation of histone H3 lysine 36 (H3K36me2) were significantly higher in SCCHN tissues than in normal epithelium. Both WHSC1 expression and H3K36me2 levels were significantly correlated with histologic grade. WHSC1 knockdown in multiple SCCHN cell lines resulted in significant growth suppression, induction of apoptosis, and delay of the cell-cycle progression. Immunoblot and immunocytochemical analyses in SCCHN cells demonstrated that WHSC1 induced H3K36me2 and H3K36me3. Microarray expression profile analysis revealed NIMA-related kinase-7 (NEK7) to be a downstream target gene of WHSC1, and chromatin immunoprecipitation (ChIP) assays showed that NEK7 was directly regulated by WHSC1 through H3K36me2. Furthermore, similar to WHSC1, NEK7 knockdown significantly reduced cell-cycle progression, indicating that NEK7 is a key player in the molecular pathway regulated by WHSC1. WHSC1 possesses oncogenic functions in SCCHN and represents a potential molecular target for the treatment of SCCHN.

Abstract 5153: Wolf-Hirschhorn syndrome candidate 1 as a potential novel therapeutic target for head and neck cancer

Abstract Background: Protein lysine methyltransferases (PKMTs) are a class of histone modifiers that have recently been reported to be mutated, amplified and overexpressed in a variety of cancers and are emerging as attractive targets for drug development. Squamous cell carcinoma of the head and neck (SCCHN) is a common malignancy with a poor prognosis. Recently, we found that WHSC1, a protein methyltransferase of the NSD family of PKMTs (NSD1, WHSC1/NSD2, WHSC1L1/NSD3), is overexpressed in 75% of SCCHN patients. In addition, the TCGA project recently reported that the NSD-family of PKMTs is altered in 26% of SCCHN patients in a mutually exclusive pattern. Given the above and the fact that an upcoming WHSC1-inhibitor is under development, we decided to further investigate the role of WHSC1 as a mediator of oncogenesis and thus as a potential novel therapeutic target for SCCHN. Methods: Cytotoxicity assays (MTTs) were performed in one HPV-positive and 3 HPV-negative SCCHN cell lines using WHSC1-specific siRNAs. Annexin V assays were performed to evaluate whether cell death was mediated through apoptosis. Cell cycle analysis by flow cytometry was also performed. Immunohistochemistry for WHSC1 and H3K36me2 was conducted in a cohort of 123 patients with SCCHN. To identify downstream targets of WHSC1, cDNA microarrays were conducted in a loss-of-function SCCHN cell line. ChIP assays were performed to assess transcriptional downstream targets directly regulated by WHSC1. Immunoprecipitation assays and mass spectrometry analysis were conducted and candidate substrates were further characterized by in vitro methyltransferase assay. Results: Knockdown of WHSC1 with WHSC1-specific siRNAs caused significant growth suppression in 4 SCCHN cell lines. Cell death was mediated partially through induction of apoptosis, as evidence by an increase of sub-G1 and annexin V-positive cells. Cell cycle analysis also revealed a significant decrease in the S-phase cells. Survival correlations of WHSC1 in a cohort of 123 SCCHN patients is currently underway. cDNA microarray analysis revealed that knockdown of WHSC1 in a SCCHN cell line was associated with downregulation of genes involved in cellular differentiation, apoptosis and mitosis, including NEK7, MAPK8 and HIPK3, which were validated with qPCR. NEK7 was further validated to be downregulated at the protein level after WHSC1 knockdown. ChIP assay for NEK7 revealed that WHSC1 directly binds to four different gene body regions of NEK7. Conclusions: Based on these data, it is possible that WHSC1 may function as a driver of oncogenesis in SCCHN, and could thus serve as a novel therapeutic target for this disease. With a WHSC1-inhibitor already under development, advancing our knowledge of the function of WHSC1 and the other NSD-PKMTs in SCCHN and other cancer types with NSD-aberrancies could accelerate the introduction of relevant inhibitors in clinical trials. Citation Format: Vassiliki Saloura, Makoto Nakakido, Houda Alachkar, Tanguy Seiwert, Mark Lingen, Ezra Cohen, Yusuke Nakamura, Ryuji Hamamoto. Wolf-Hirschhorn syndrome candidate 1 as a potential novel therapeutic target for head and neck cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5153. doi:10.1158/1538-7445.AM2014-5153

Histone H3 lysine 36 methyltransferase Whsc1 promotes the association of Runx2 and p300 in the activation of bone-related genes.

The orchestration of histone modifiers is required to establish the epigenomic status that regulates gene expression during development. Whsc1 (Wolf-Hirschhorn Syndrome candidate 1), a histone H3 lysine 36 (H3K36) trimethyltransferase, is one of the major genes associated with Wolf-Hirshhorn syndrome, which is characterized by skeletal abnormalities. However, the role of Whsc1 in skeletal development remains unclear. Here, we show that Whsc1 regulates gene expression through Runt-related transcription factor (Runx) 2, a transcription factor central to bone development, and p300, a histone acetyltransferase, to promote bone differentiation. Whsc1 −/− embryos exhibited defects in ossification in the occipital bone and sternum. Whsc1 knockdown in pre-osteoblast cells perturbed histone modification patterns in bone-related genes and led to defects in bone differentiation. Whsc1 increased the association of p300 with Runx2, activating the bone-related genes Osteopontin (Opn) and Collagen type Ia (Col1a1), and Whsc1 suppressed the overactivation of these genes via H3K36 trimethylation. Our results suggest that Whsc1 fine-tunes the expression of bone-related genes by acting as a modulator in balancing H3K36 trimethylation and histone acetylation. Our results provide novel insight into the mechanisms by which this histone methyltransferase regulates gene expression.

Lung tumor-associated dendritic cell-derived resistin promoted cancer progression by increasing Wolf–Hirschhorn syndrome candidate 1/Twist pathway

The interaction between tumors and their microenvironments leads to a vicious cycle, which strengthens both immune suppression and cancer progression. The present study demonstrates for the first time that tumor-associated dendritic cells (TADCs) are a source of resistin, which is responsible for increasing lung cancer epithelial-to-mesenchymal transition. In addition, large amounts of resistin in the condition medium (CM) of TADCs increase cell migration and invasion, as well as the osteolytic bone metastatic properties of lung cancer cells. Neutralization of resistin from TADC-CM prevents the advanced malignancy-inducing features of TADC-CM. Significantly elevated levels of resistin have been observed in mice transplanted with lung cancer cells, tumor-infiltrating CD11c(+) DCs in human lung cancer samples and lung cancer patients' sera. Induction of lung cancer progression by TADC-derived resistin is associated with increased expression of Wolf-Hirschhorn syndrome candidate 1 (WHSC1), a histone methyltransferase. Resistin-induced WHSC1 increases the dimethylation of histone 3 at lysine 36 and decreases the trimethylation of histone 3 at lysine 27 on the promoter of Twist, resulting in an enhancement of the expression of Twist. Knockdown of WHSC1 by small interfering RNA transfection significantly decreases resistin-mediated cancer progression by decreasing the upregulation of Twist, suggesting that WHSC1 plays a critical role in the regulation of Twist by epigenetic modification. Furthermore, mice that received antiresistin antibodies showed a decreased incidence of cancer development and metastasis. These findings suggest that TADC-derived resistin may be a novel candidate in promoting the development of lung cancer.

WHSC1 links transcription elongation to HIRA-mediated histone H3.3 deposition

Actively transcribed genes are enriched with the histone variant H3.3. Although H3.3 deposition has been linked to transcription, mechanisms controlling this process remain elusive. We investigated the role of the histone methyltransferase Wolf-Hirschhorn syndrome candidate 1 (WHSC1) (NSD2/MMSET) in H3.3 deposition into interferon (IFN) response genes. IFN treatment triggered robust H3.3 incorporation into activated genes, which continued even after cessation of transcription. Likewise, UV radiation caused H3.3 deposition in UV-activated genes. However, in Whsc1(-/-) cells IFN- or UV-triggered H3.3 deposition was absent, along with a marked reduction in IFN- or UV-induced transcription. We found that WHSC1 interacted with the bromodomain protein 4 (BRD4) and the positive transcription elongation factor b (P-TEFb) and facilitated transcriptional elongation. WHSC1 also associated with HIRA, the H3.3-specific histone chaperone, independent of BRD4 and P-TEFb. WHSC1 and HIRA co-occupied IFN-stimulated genes and supported prolonged H3.3 incorporation, leaving a lasting transcriptional mark. Our results reveal a previously unrecognized role of WHSC1, which links transcriptional elongation and H3.3 deposition into activated genes through two molecularly distinct pathways.

Abstract 2979: Wolf-Hirschhorn syndrome candidate 1 plays an important role in the pathogenesis of head and neck cancer.

Abstract Wolf-Hirschhorn syndrome candidate 1 (WHSC1) is a histone lysine methyltransferase (HMT) which was initially found to be deregulated in a subset of multiple myeloma cells with the t(4;14) (p16;q32) translocation and poor prognosis. Recently, our group as well as others found that WHSC1 was overexpressed in a variety of tumors, including lung and esophageal squamous cell carcinomas, with low or no expression in normal tissues except the testis. Long-term survival of patients with squamous cell carcinoma of the head and neck (SCCHN) remains poor and thus new therapeutic strategies are urgently needed. Epigenetic alterations have mainly been studied as a potential mechanism of progression from dysplasia to squamous cell carcinoma. Given the promising potential of HMTs as novel drug targets and the aforementioned interesting expression patterns, we further investigated the role of WHSC1 in carcinogenesis through immunohistochemical analysis (IHC) using tissue microarrays of 123 locoregionally advanced SCCHN patients, and 6 samples of fibroepithelial hyperplasia lesions obtained from oral biopsies. We classified its expression levels with a scoring scale of 0 (no expression), +1 (mild), +2 (moderate) or +3 (strong). WHSC1 expression in the nucleus and cytoplasm was moderate (score +2, 37%) or strong (score +3, 38%) in 75% of SCCHN samples, while 25% showed mild (score +1, 24%) or no (score 0, 1%) expression. WHSC1 staining of normal epithelial regions revealed an exclusively nuclear expression pattern in the basal and parabasal layers. This is consistent with reports suggesting a physiologic role of the Wnt-pathway, which was recently found to be induced by WHSC1, in the differentiation process of the squamous epithelium. We also performed Western blot analysis of WHSC1 in 8 SCCHN cells lines, 3 control fibroblast cell lines and 1 keratinocyte cell line. We observed moderate to strong overexpression of WHSC1 in 4 of 8 SCCHN cell lines, while no expression was detected in the normal keratinocytes or the fibroblast cell lines. These findings support a potential role of the WHSC1 pathway in the pathogenesis of SCCHN and provides a new therapeutic avenue for drug discovery in this fatal disease. Citation Format: Vassiliki Saloura, Hyun-Soo Cho, Mark Lingen, Terri Li, Can Gong, Seiwert Tanguy, Ezra Cohen, Ryuji Hamamoto, Yusuke Nakamura. Wolf-Hirschhorn syndrome candidate 1 plays an important role in the pathogenesis of head and neck cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2979. doi:10.1158/1538-7445.AM2013-2979

Abstract 2964: Epigenetic mechanisms of transformation by WHSC1L1 in breast cancer cells with the 8p11-p12 amplicon.

Abstract An amplified region of chromosome 8, the 8p11-p12 amplicon, is observed in ∼15% of breast cancers. This region contains 55 genes, of which 22 have been identified as candidate oncogenes based on higher than normal copy number and associated elevated expression levels in breast tumor cells compared to normal breast epithelial cells. Our lab previously identified the short isoform of one amplicon gene, Wolf Hirschhorn Syndrome Candidate 1-Like 1 (WHSC1L1), to be a potent inducer of transformation when over expressed in MCF10A breast epithelial cells in vitro. WHSC1L1 is a member of the NSD family of methyltransferases, and has been shown to methylate lysines 4, 27, and 36 of the histone H3 subunit, however, the potently transforming short isoform (WHshort) lacks the catalytic SET domain. Our lab has shown that over expression of WHshort in the immortalized human breast epithelial cell line MCF10A causes a significant increase in proliferation in insulin-free culture conditions, and the ability to form colonies in soft agar. In SUM44 cells, an ER+ breast cancer cell line harboring the 8p11-p12 amplicon, shRNA knockdown of WHSC1L1 resulted in growth arrest. The WHSC1L1 family member NSD1 has been shown to act as an oncogene in acute myelogenous leukemia by blocking differentiation in developing lymphocytes through H3K36me3-mediated inhibition of the polycomb repressor complex, resulting in constitutive activation of the HOXA gene family. While WHSC1L1 may work by a similar mechanism, little is known about the specific substrates of WHSC1L1, or even whether its oncogenic activity is caused by histone methylation changes. Knockdown by shRNA of WHSC1L1 in SUM44 cells resulted in a significant reduction in transcript levels of several genes important in breast cancer cell proliferation, including ESR1, MYCN (amplified in SUM-44), ERBB3, and ERBB4. Interestingly, Carroll et al. recently reported that ER+ breast tumors can be stratified by gene expression signature into poor outcome and good outcome groups, and that ER-alpha binding site patterns on chromatin were altered in the two groups. In this study, WHSC1L1 was part of the ER-regulated gene signature of the poor outcome group. Therefore, we hypothesize that WHSC1L1 may influence histone H3K36me3 marks at specific gene loci, leading to altered ER-alpha binding that is associated with the expression signatures of poor-outcome ER+ breast cancers. To investigate chromatin changes as a result of changes in WHSC1L1 expression, we have accomplished shRNA knockdown of WHSC1L1 in SUM44 cells using GIPZ and TRIPZ lentiviral vectors. Using these cell lines, we are investigating changes in H3K36me3 marks and ER-alpha binding patterns by chromatin immunoprecipitation followed by sequencing (ChIP-seq). These experiments will help to elucidate the interaction of WHSC1L1 and ER in regulated gene expression in breast cancer cells with the 8p11 amplicon. Citation Format: Jonathan C. Irish, Gang Liu, Stephen P. Ethier. Epigenetic mechanisms of transformation by WHSC1L1 in breast cancer cells with the 8p11-p12 amplicon. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2964. doi:10.1158/1538-7445.AM2013-2964

WHSC1 (Wolf-Hirschhorn syndrome candidate 1)

Review on WHSC1 (Wolf-Hirschhorn syndrome candidate 1), with data on DNA, on the protein encoded, and where the gene is implicated.

The histone methyltransferase Wolf–Hirschhorn syndrome candidate 1‐like 1 (WHSC1L1) is involved in human carcinogenesis

Histone lysine methylation plays a fundamental role in chromatin organization. Although a set of histone methyltransferases have been identified and biochemically characterized, the pathological roles of their dysfunction in human cancers are still not well understood. In this study, we demonstrate important roles of WHSC1L1 in human carcinogenesis. Expression levels of WHSC1L1 transcript were significantly elevated in various human cancers including bladder carcinoma. Immunohistochemical analysis of bladder, lung, and liver cancers confirmed overexpression of WHSC1L1. WHSC1L1-specific small interfering RNAs significantly knocked down its expression and resulted in suppression of proliferation of bladder and lung cancer cell lines. WHSC1L1 knockdown induced cell cycle arrest at the G(2)/M phase followed by multinucleation of cancer cells. Expression profile analysis using Affymetrix GeneChip(®) showed that WHSC1L1 affected the expression of a number of genes including CCNG1 and NEK7, which are known to play crucial roles in the cell cycle progression at mitosis. As WHSC1L1 expression is significantly low in various normal tissues including vital organs, WHSC1L1 could be a good candidate molecule for development of novel treatment for various types of cancer.

A microdeletion proximal of the critical deletion region is associated with mild Wolf–Hirschhorn syndrome

It is generally accepted that the facial phenotype of Wolf-Hirschhorn syndrome is caused by deletions of either Wolf-Hirschhorn critical regions 1 or 2 (WHSCR 1-2). Here, we identify a 432 kb deletion located 600 kb proximal to both WHSCR1-2 in a patient with a WHS facial phenotype. Seven genes are underlying this deletion region including FAM193a, ADD1, NOP14, GRK4, MFSD10, SH3BP2, TNIP2. The clinical diagnosis of WHS facial phenotype was confirmed by 3D facial analysis using dense surface modeling. Our results suggest that the WHSCR1-2 flanking sequence contributes directly or indirectly to the severity of WHS. Sequencing the Wolf-Hirschhorn syndrome candidate 1 and 2 genes did not reveal any mutations. Long range position effects of the deletion that could influence gene expression within the WHSCR were excluded in EBV cell lines derived from patient lymphoblasts. We hypothesize that either (1) this locus harbors regulatory sequences which affect gene expression in the WHSCR1-2 in a defined temporal and spatial developmental window or (2) that this locus is additive to deletions of WHSCR1-2 increasing the phenotypic expression.

The Histone Methytransferase MMSET Regulates Class-Switch Recombination

Abstract 691▪▪This icon denotes a clinically relevant abstractMalignant plasma cells in multiple myeloma (MM) patients display a variety of recurrent genetic abnormalities. In this regard, tumor cells in approximately 15% of all MM patients will exhibit a translocation involving the immunoglobulin (Ig) heavy chain locus at 14q32 and the short arm of chromosome 4. The breakpoint on chromosome 4 (4p16) frequently results in overexpression of FGFR3 and/or full-length or truncated versions of the multiple myeloma SET domain protein, MMSET. MM patients with t(4;14) translocations are considered to have high-risk disease. MMSET has been shown to have histone methyltransferase activity and we have recently shown that this protein plays a pivotal role in DNA repair and maintenance of genetic stability (1). Thus, dysregulation of MMSET may result in aberrant responses to DNA damage, which may be related to the poor prognosis of MM patients with t(4;14) translocations. The MMSET gene is also known as the Wolf-Hirschhorn syndrome candidate (WHSC1) gene. Expression of the WHSC1 gene is uniformly misregulated due to haploinsufficiency in patients with Wolf-Hirschhorn syndrome (WHS) resulting in characteristic facial features and developmental disorders. Of great interest, WHS patients also display significant antibody deficiencies and IgG and IgA deficiencies are particularly frequent. Currently, the underlying cause of antibody deficiencies in WHS patients remains unknown. However, our recent studies have shown that a robust DNA repair process in germinal center B cells is required for fertile antibody maturation processes (2). This observation, taken together with our recent discover that MMSET regulates the recruitment of 53BP1 to sites of DNA damage through its histone methyltransferase activity during DNA damage repair (1), suggested to us the hypothesis that MMSET may also be critically involved in Ig gene maturation, particularly as it concerns class switch recombination, a process known to result from double strand DNA breaks and subsequent effective DNA repair. By using shRNA knockdown technology in murine lymphoma cell line CH12F3 cells, which can be specifically induced to switch from IgM to IgA expression ex vivo by CD40 ligand stimulation in the presence of IL-4 and TGFb, we clearly demonstrate that downregulation of MMSET expression by shRNA significantly impaired class switch recombination from IgM to IgA. While it plays no detectable roles in cell viability, proliferation, or apoptosis, we found that MMSET is important for histone methylation at H3K36 and H4K20 sites of the Igh loci, which in turn modulate the recruitment of 53BP1 to the Igh loci as well as the transcription of the Igh switch regions, leading to defective class switch recombination. Further DNA sequence analysis of post-switched Sm-Sa junctions from MMSET compromised cells showed a significant increase in microhomology suggesting that homologous recombination (HR) repair is alternatively used as a compensatory mechanism during DNA repair of the switch region when MMSET is absent. Our results suggest that defective CSR caused by MMSET deficiency may underpin the antibody deficiency phenotype in WHS patients. Furthermore, our results showing that MMSET expression dose dictates the usage choice between two competing DNA repair pathways, i.e., error-prone non-homologous end joining (NHEJ) and error-free HR, may also suggest that MMSET overexpression in MM may favor usage of the NHEJ pathway therefore leading to more error-prone DNA repair and possibly additional genetic damage. Disclosures:No relevant conflicts of interest to declare.

Histone Lysine Methyltransferase Wolf-Hirschhorn Syndrome Candidate 1 Is Involved in Human Carcinogenesis through Regulation of the Wnt Pathway

A number of histone methyltransferases have been identified and biochemically characterized, but the pathologic roles of their dysfunction in human diseases like cancer are not well understood. Here, we demonstrate that Wolf-Hirschhorn syndrome candidate 1 (WHSC1) plays important roles in human carcinogenesis. Transcriptional levels of this gene are significantly elevated in various types of cancer including bladder and lung cancers. Immunohistochemical analysis using a number of clinical tissues confirmed significant up-regulation of WHSC1 expression in bladder and lung cancer cells at the protein level. Treatment of cancer cell lines with small interfering RNA targeting WHSC1 significantly knocked down its expression and resulted in the suppression of proliferation. Cell cycle analysis by flow cytometry indicated that knockdown of WHSC1 decreased the cell population of cancer cells at the S phase while increasing that at the G2/M phase. WHSC1 interacts with some proteins related to the WNT pathway including β-catenin and transcriptionally regulates CCND1, the target gene of the β-catenin/Tcf-4 complex, through histone H3 at lysine 36 trimethylation. This is a novel mechanism for WNT pathway dysregulation in human carcinogenesis, mediated by the epigenetic regulation of histone H3. Because expression levels of WHSC1 are significantly low in most normal tissue types, it should be feasible to develop specific and selective inhibitors targeting the enzyme as antitumor agents that have a minimal risk of adverse reaction.

Wolf–Hirschhorn syndrome candidate 1 is involved in the cellular response to DNA damage

Wolf-Hirschhorn syndrome (WHS) is a malformation syndrome associated with growth retardation, mental retardation, and immunodeficiency resulting from a hemizygous deletion of the short arm of chromosome 4, called the WHS critical region (WHSC). The WHSC1 gene is located in this region, and its loss is believed to be responsible for a number of WHS characteristics. We identified WHSC1 in a genetic screen for genes involved in responding to replication stress, linking Wolf-Hirschhorn syndrome to the DNA damage response (DDR). Here, we report that the WHSC1 protein is a member of the DDR pathway. WHSC1 localizes to sites of DNA damage and replication stress and is required for resistance to many DNA-damaging and replication stress-inducing agents. Through its SET domain, WHSC1 regulates the methylation status of the histone H4 K20 residue and is required for the recruitment of 53BP1 to sites of DNA damage. We propose that Wolf-Hirschhorn syndrome results from a defect in the DDR.

Abstract 4819: Wolf-Hirschhorn syndrome candidate 1, a histone lysine methyltransferase, is involved in human carcinogenesis

Abstract A number of histone methyltransferases have been identified and biochemically characterized, but the pathological roles of their dysfunction in human diseases like cancer are not well understood. Here, we demonstrate that Wolf-Hirschhorn syndrome candidate 1 (WHSC1) plays an important role in human carcinogenesis. Transcriptional levels of this gene are significantly elevated in various types of cancer including bladder and lung cancers. Immunohistochemical analysis using a number of clinical tissues confirmed significant up-regulation of WHSC1 expression in bladder and lung cancer cells at the protein level. Treatment of cancer cell lines with small interfering RNAs targeting WHSC1 significantly knocked down its expression and resulted in the suppression of proliferation. Moreover, knockdown of WHSC1 decreased the cell population of cancer cells at S phase and increased that at G2/M phase through the regulation of genes involved in the Wnt signaling pathway. Furthermore, we found WHSC1 to be interacted with some proteins related to the Wnt pathway including β-catenin. As expression levels of WHSC1 are significantly low in normal tissues, it may be feasible to develop the inhibitors targeting these enzymes as anti-tumor agents which have a minimal risk of adverse reaction. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4819. doi:10.1158/1538-7445.AM2011-4819