Steven A. Johnsen

The histone H2B-specific ubiquitin ligase RNF20/hBRE1 acts as a putative tumor suppressor through selective regulation of gene expression

Histone monoubiquitylation is implicated in critical regulatory processes. We explored the roles of histone H2B ubiquitylation in human cells by reducing the expression of hBRE1/RNF20, the major H2B-specific E3 ubiquitin ligase. While H2B ubiquitylation is broadly associated with transcribed genes, only a subset of genes was transcriptionally affected by RNF20 depletion and abrogation of H2B ubiquitylation. Gene expression dependent on RNF20 includes histones H2A and H2B and the p53 tumor suppressor. In contrast, RNF20 suppresses the expression of several proto-oncogenes, which reside preferentially in closed chromatin and are modestly transcribed despite bearing marks usually associated with high transcription rates. Remarkably, RNF20 depletion augmented the transcriptional effects of epidermal growth factor (EGF), increased cell migration, and elicited transformation and tumorigenesis. Furthermore, frequent RNF20 promoter hypermethylation was observed in tumors. RNF20 may thus be a putative tumor suppressor, acting through selective regulation of a distinct subset of genes.

CDK9 directs H2B monoubiquitination and controls replication‐dependent histone mRNA 3′‐end processing

Post‐translational histone modifications have essential roles in controlling nuclear processes; however, the specific mechanisms regulating these modifications and their combinatorial activities remain elusive. Cyclin‐dependent kinase 9 (CDK9) regulates gene expression by phosphorylating transcriptional regulatory proteins, including the RNA polymerase II carboxy‐terminal domain. Here, we show that CDK9 activity is essential for maintaining global and gene‐associated levels of histone H2B monoubiquitination (H2Bub1). Furthermore, CDK9 activity and H2Bub1 help to maintain correct replication‐dependent histone messenger RNA (mRNA) 3′‐end processing. CDK9 knockdown consistently resulted in inefficient recognition of the correct mRNA 3′‐end cleavage site and led to increased read‐through of RNA polymerase II to an alternative downstream polyadenylation signal. Thus, CDK9 acts to integrate phosphorylation during transcription with chromatin modifications to control co‐transcriptional histone mRNA processing.

The Histone H2B Monoubiquitination Regulatory Pathway Is Required for Differentiation of Multipotent Stem Cells

Extensive changes in posttranslational histone modifications accompany the rewiring of the transcriptional program during stem cell differentiation. However, the mechanisms controlling the changes in specific chromatin modifications and their function during differentiation remain only poorly understood. We show that histone H2B monoubiquitination (H2Bub1) significantly increases during differentiation of human mesenchymal stem cells (hMSCs) and various lineage-committed precursor cells and in diverse organisms. Furthermore, the H2B ubiquitin ligase RNF40 is required for the induction of differentiation markers and transcriptional reprogramming of hMSCs. This function is dependent upon CDK9 and the WAC adaptor protein, which are required for H2B monoubiquitination. Finally, we show that RNF40 is required for the resolution of the H3K4me3/H3K27me3 bivalent poised state on lineage-specific genes during the transition from an inactive to an active chromatin conformation. Thus, these data indicate that H2Bub1 is required for maintaining multipotency of hMSCs and plays a central role in controlling stem cell differentiation.

Estrogen-Dependent Gene Transcription in Human Breast Cancer Cells Relies upon Proteasome-Dependent Monoubiquitination of Histone H2B

The estrogen receptor-α (ERα) determines the phenotype of breast cancers where it serves as a positive prognostic indicator. ERα is a well-established target for breast cancer therapy, but strategies to target its function remain of interest to address therapeutic resistance and further improve treatment. Recent findings indicate that proteasome inhibition can regulate estrogen-induced transcription, but how ERα function might be regulated was uncertain. In this study, we investigated the transcriptome-wide effects of the proteasome inhibitor bortezomib on estrogen-regulated transcription in MCF7 human breast cancer cells and showed that bortezomib caused a specific global decrease in estrogen-induced gene expression. This effect was specific because gene expression induced by the glucocorticoid receptor was unaffected by bortezomib. Surprisingly, we observed no changes in ERα recruitment or assembly of its transcriptional activation complex on ERα target genes. Instead, we found that proteasome inhibition caused a global decrease in histone H2B monoubiquitination (H2Bub1), leading to transcriptional elongation defects on estrogen target genes and to decreased chromatin dynamics overall. In confirming the functional significance of this link, we showed that RNA interference-mediated knockdown of the H2B ubiquitin ligase RNF40 decreased ERα-induced gene transcription. Surprisingly, RNF40 knockdown also supported estrogen-independent cell proliferation and activation of cell survival signaling pathways. Most importantly, we found that H2Bub1 levels decrease during tumor progression. H2Bub1 was abundant in normal mammary epithelium and benign breast tumors but absent in most malignant and metastatic breast cancers. Taken together, our findings show how ERα activity is blunted by bortezomib treatment as a result of reducing the downstream ubiquitin-dependent function of H2Bub1. In supporting a tumor suppressor role for H2Bub1 in breast cancer, our findings offer a rational basis to pursue H2Bub1-based therapies for future management of breast cancer.

Role of TIEG1 in biological processes and disease states

A novel TGFβ Inducible Early Gene‐1 (TIEG1) was discovered in human osteoblast (OB) cells by our laboratory. Over the past decade, a handful of laboratories have revealed a multitude of organismic, cellular, and molecular functions of this gene. TIEG1 is now classified as a member of the 3 zinc finger family of Krüppel‐like transcription factors (KLF10). Other closely related factors [TIEG2 (KLF11) and TIEG3/TIEG2b] have been reported and are briefly compared. As described in this review, TIEG1 is shown to play a role in regulating estrogen and TGFβ actions, the latter through the Smad signaling pathway. In both cases, TIEG1 acts as an inducer or repressor of gene transcription to enhance the TGFβ/Smad pathway, as well at other signaling pathways, to regulate cell proliferation, differentiation, and apoptosis. This review outlines TIEG1s molecular functions and roles in skeletal disease (osteopenia/osteoporosis), heart disease (hypertrophic cardiomyopathy), and cancer (breast and prostate). J. Cell. Biochem. 102: 539–548, 2007.

Bromodomain protein BRD4 is required for estrogen receptor-dependent enhancer activation and gene transcription.

SUMMARY The estrogen receptor α (ERα) controls cell proliferation and tumorigenesis by recruiting various cofactors to estrogen response elements (EREs) to control gene transcription. A deeper understanding of these transcriptional mechanisms may uncover therapeutic targets for ERα-dependent cancers. We show that BRD4 regulates ERα-induced gene expression by affecting elongation-associated phosphorylation of RNA polymerase II (RNAPII) and histone H2B monoubiquitination. Consistently, BRD4 activity is required for proliferation of ER+ breast and endometrial cancer cells and uterine growth in mice. Genome-wide studies revealed an enrichment of BRD4 on transcriptional start sites of active genes and a requirement of BRD4 for H2B monoubiquitination in the transcribed region of estrogen-responsive genes. Importantly, we demonstrate that BRD4 occupancy on distal EREs enriched for H3K27ac is required for recruitment and elongation of RNAPII on EREs and the production of ERα-dependent enhancer RNAs. These results uncover BRD4 as a central regulator of ERα function and potential therapeutic target.

The enigmatic role of H2Bub1 in cancer

The post‐translational modification of histone proteins plays an important role in controlling cell fate by directing essentially all DNA‐associated nuclear processes. Misregulation and mutation of histone modifying enzymes is a hallmark of tumorigenesis. However, how these different epigenetic modifications lead to tumor initiation and/or progression remains poorly understood. Recent studies have uncovered a potential tumor suppressor role for histone H2B monoubiquitination (H2Bub1). Like many other histone modifications, H2Bub1 has diverse functions and plays roles both in transcriptional activation and repression as well as in controlling mRNA processing and directing DNA repair processes. Notably, H2Bub1 has been linked to transcriptional elongation and is preferentially found in the transcribed region of active genes. Its activity is intimately connected to active transcription and the transcriptional elongation regulatory protein cyclin‐dependent kinase‐9 (CDK9) and the facilitates chromatin transcription (FACT) complex. This review provides an overview of the current understanding of H2Bub1 function in mammalian systems with a particular emphasis on its role in cancer and potential options for exploiting this knowledge for the treatment of cancer.

The H2B ubiquitin ligase RNF40 cooperates with SUPT16H to induce dynamic changes in chromatin structure during DNA double-strand break repair

Many anticancer therapies function largely by inducing DNA double-strand breaks (DSBs) or altering the ability of cancer cells to repair them. Proper and timely DNA repair requires dynamic changes in chromatin assembly and disassembly characterized by histone H3 lysine 56 acetylation (H3K56ac) and phosphorylation of the variant histone H2AX (γH2AX). Similarly, histone H2B monoubiquitination (H2Bub1) functions in DNA repair, but its role in controlling dynamic changes in chromatin structure following DSBs and the histone chaperone complexes involved remain unknown. Therefore, we investigated the role of the H2B ubiquitin ligase RNF40 in the DSB response. We show that RNF40 depletion results in sustained H2AX phosphorylation and a decrease in rapid cell cycle checkpoint activation. Furthermore, RNF40 knockdown resulted in decreased H3K56ac and decreased recruitment of the facilitates chromatin transcription (FACT) complex to chromatin following DSB. Knockdown of the FACT component suppressor of Ty homolog-16 (SUPT16H) phenocopied the effects of RNF40 knockdown on both γH2AX and H3K56ac following DSB induction. Consistently, both RNF40 and SUPT16H were required for proper DNA end resection and timely DNA repair, suggesting that H2Bub1 and FACT cooperate to increase chromatin dynamics, which facilitates proper checkpoint activation and timely DNA repair. These results provide important mechanistic insights into the tumor suppressor function of H2Bub1 and provide a rational basis for pursuing H2Bub1-based therapies in conjunction with traditional chemo- and radiotherapy.

Selection of Patients for Long-term Surveillance With Digital Dermoscopy by Assessment of Melanoma Risk Factors

OBJECTIVE To identify patients at increased melanoma risk who benefit from long-term surveillance with digital dermoscopy. DESIGN Prospective, nonrandomized, observational study. SETTING University-based surveillance program. PARTICIPANTS Six hundred eighty-eight patients prospectively categorized into defined melanoma risk groups and followed up (mean, 44.3 months) by clinical examinations, dermoscopy, and, for atypical nevi, sequential digital dermoscopy. MAIN OUTCOME MEASURE Association between patient risk factors and detection of melanomas. RESULTS Odds ratios from a multivariate logistic regression analysis indicated a highly increased melanoma risk for patients with familial atypical mole and multiple melanoma (FAMMM) syndrome, atypical mole syndrome (AMS), or previous melanoma. Each digitally documented atypical lesion (range, 1-17 lesions per patient) denoted a significant 10% increase in melanoma risk. Patients with higher melanoma risk (1) showed a higher percentage of melanomas detected by digital dermoscopy (FAMMM syndrome group, 50%; AMS group, 22%), (2) more often developed multiple melanomas within shorter intervals, and (3) showed a ratio of melanoma to benign results for lesions excised because of dynamic changes of 1:15 (AMS group) or 1:4 (FAMMM syndrome group). Melanomas detected by digital dermoscopy were significantly thinner (0.41 mm in mean Breslow thickness) compared with melanomas detected by other means (0.62 mm; P = .04). CONCLUSIONS We suggest an individualized surveillance plan, with digital dermoscopy performed at follow-up intervals of 3 months for patients with FAMMM syndrome and 6 to 12 months (depending on additional risk factors) for those with AMS. Patients with multiple common nevi and no additional risk factors had no benefit from sequential digital dermoscopy.

Stem-cell-like properties and epithelial plasticity arise as stable traits after transient Twist1 activation.

Master regulators of the epithelial-mesenchymal transition such as Twist1 and Snail1 have been implicated in invasiveness and the generation of cancer stem cells, but their persistent activity inhibits stem-cell-like properties and the outgrowth of disseminated cancer cells into macroscopic metastases. Here, we show that Twist1 activation primes a subset of mammary epithelial cells for stem-cell-like properties, which only emerge and stably persist following Twist1 deactivation. Consequently, when cells undergo a mesenchymal-epithelial transition (MET), they do not return to their original epithelial cell state, evidenced by acquisition of invasive growth behavior and a distinct gene expression profile. These data provide an explanation for how transient Twist1 activation may promote all steps of the metastatic cascade; i.e., invasion, dissemination, and metastatic outgrowth at distant sites.