Faculty Bibliography

The past decade brought a revolution in understanding of the structure, topology and disease-inducing lesions of RNA and DNA, fueled by unprecedented progress in next-generation sequencing. This technological revolution has also affected understanding of the epigenome and has provided unique opportunities for the analysis of DNA and histone modifications, as well as the first map of the non-protein-coding genome and three-dimensional (3D) chromosomal interactions. Overall, these advances have facilitated studies that combine genetic, transcriptomics and epigenomics data to address a wide range of issues ranging from understanding the role of the epigenome in development to targeting the transcription of noncoding genes in human cancer. Here we describe recent insights into epigenetic dysregulation characteristic of the malignant differentiation of blood stem cells based on studies of alterations that affect epigenetic complexes, enhancers, chromatin, long noncoding RNAs (lncRNAs), RNA splicing, nuclear topology and the 3D conformation of chromatin.

Cell-specific gene expression programs rely on the orchestrated function of transcription factors, co-activators, the transcriptional machinery and non-coding RNA elements at enhancer and promoter elements. Mediator is a large co-activator complex that bridges promoters with transcription factors bound to cell-specific enhancers. Here we describe Med12, a member of the Mediator complex kinase module, as an indispensable regulator of hematopoietic stem cell (HSC) maintenance and differentiation. Conditional deletion of Med12 rapidly exhausts adult bone marrow HSCs leading to lethality. Conversely, HSC-specific deletion of other members of the Mediator complex kinase module, Med13, CyclinC and CDK8 are dispensable for HSC function. We also show that Med12 is an enhancer element that co-localizes with essential hematopoietic transcription factors and enhancer-specific histone marks. In this context, we demonstrate that Med12 loss in HSCs results in rapid dysregulation of stemness signatures, as well as failure of enhancer activation by depletion of H3K27Ac. Finally, we find that Med12 promotes stabilization of p300 at enhancers, thereby contributing to the preservation of HSC homeostasis. Collectively, our data describes a novel, kinase-independent function for Med12 in the regulation of HCS enhancer elements and the maintenance of normal stem cell function. As a result, we believe that alterations in the Med12-dependent regulation of HSC enhancers may contribute to malignant transformation and disease

Mutations in the isocitrate dehydrogenase-1 gene (IDH1) are common drivers of acute myeloid leukemia (AML) but their mechanism is not fully understood. It is thought that IDH1 mutants act by inhibiting TET2 to alter DNA methylation, but there are significant unexplained clinical differences between IDH1- and TET2-mutant diseases. We have discovered that mice expressing endogenous mutant IDH1 have reduced numbers of hematopoietic stem cells (HSCs), in contrast to Tet2 knockout (TET2-KO) mice. Mutant IDH1 downregulates the DNA damage (DD) sensor ATM by altering histone methylation, leading to impaired DNA repair, increased sensitivity to DD, and reduced HSC self-renewal, independent of TET2. ATM expression is also decreased in human IDH1-mutated AML. These findings may have implications for treatment of IDH-mutant leukemia.

V(D)J recombination relies on the presence of proximal enhancers that activate the antigen receptor (AgR) loci in a lineage- and stage-specific manner. Unexpectedly, we find that both active and inactive AgR enhancers cooperate to disseminate their effects in a localized and long-range manner. Here, we demonstrate the importance of short-range contacts between active enhancers that constitute an Igk super-enhancer in B cells. Deletion of one element reduces the interaction frequency between other enhancers in the hub, which compromises the transcriptional output of each component. Furthermore, we establish that, in T cells, long-range contact and cooperation between the inactive Igk enhancer MiEkappa and the active Tcrb enhancer Ebeta alters enrichment of CBFbeta binding in a manner that impacts Tcrb recombination. These findings underline the complexities of enhancer regulation and point to a role for localized and long-range enhancer-sharing between active and inactive elements in lineage- and stage-specific control.

Basal cell carcinoma (BCC) of the skin is the most common malignant neoplasm in humans. BCC is primarily driven by the Sonic Hedgehog (Hh) pathway. However, its phenotypic variation remains unexplained. Our genetic profiling of 293 BCCs found the highest mutation rate in cancer (65 mutations/Mb). Eighty-five percent of the BCCs harbored mutations in Hh pathway genes (PTCH1, 73% or SMO, 20% (P = 6.6 x 10-8) and SUFU, 8%) and in TP53 (61%). However, 85% of the BCCs also harbored additional driver mutations in other cancer-related genes. We observed recurrent mutations in MYCN (30%), PPP6C (15%), STK19 (10%), LATS1 (8%), ERBB2 (4%), PIK3CA (2%), and NRAS, KRAS or HRAS (2%), and loss-of-function and deleterious missense mutations were present in PTPN14 (23%), RB1 (8%) and FBXW7 (5%). Consistent with the mutational profiles, N-Myc and Hippo-YAP pathway target genes were upregulated. Functional analysis of the mutations in MYCN, PTPN14 and LATS1 suggested their potential relevance in BCC tumorigenesis.

Aberrant DNA methylation is a characteristic feature of cancer including blood malignancies. Mutations in the DNA methylation regulators DNMT3A, TET1/2 and IDH1/2 are recurrent in leukemia and lymphoma. Specific and distinct DNA methylation patterns characterize subtypes of AML and lymphoma. Regulatory regions such as promoter CpG islands, CpG shores and enhancers show changes in methylation during transformation. However, the reported poor correlation between changes in methylation and gene expression in many mouse models and human studies reflects the complexity in the precise molecular mechanism for why aberrant DNA methylation promotes malignancies. This review will summarize current concepts regarding the mechanisms behind aberrant DNA methylation in hematopoietic malignancy and discuss its importance in cancer prognosis, tumor heterogeneity and relapse.

RATIONALE: Fatty acid oxidation is transcriptionally regulated by peroxisome proliferator-activated receptor (PPAR)alpha and under normal conditions accounts for 70% of cardiac ATP content. Reduced Ppara expression during sepsis and heart failure leads to reduced fatty acid oxidation and myocardial energy deficiency. Many of the transcriptional regulators of Ppara are unknown. OBJECTIVE: To determine the role of Kruppel-like factor 5 (KLF5) in transcriptional regulation of Ppara. METHODS AND RESULTS: We discovered that KLF5 activates Ppara gene expression via direct promoter binding. This is blocked in hearts of septic mice by c-Jun, which binds an overlapping site on the Ppara promoter and reduces transcription. We generated cardiac myocyte-specific Klf5 knockout mice that showed reduced expression of cardiac Ppara and its downstream fatty acid metabolism-related targets. These changes were associated with reduced cardiac fatty acid oxidation, ATP levels, increased triglyceride accumulation and cardiac dysfunction. Diabetic mice showed parallel changes in cardiac Klf5 and Ppara expression levels. CONCLUSIONS: Cardiac myocyte KLF5 is a transcriptional regulator of Ppara and cardiac energetics.