Faculty Bibliography

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.

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.

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.

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.

The cohesin complex (consisting of Rad21, Smc1a, Smc3, and Stag2 proteins) is critically important for proper sister chromatid separation during mitosis. Mutations in the cohesin complex were recently identified in a variety of human malignancies including acute myeloid leukemia (AML). To address the potential tumor-suppressive function of cohesin in vivo, we generated a series of shRNA mouse models in which endogenous cohesin can be silenced inducibly. Notably, silencing of cohesin complex members did not have a deleterious effect on cell viability. Furthermore, knockdown of cohesin led to gain of replating capacity of mouse hematopoietic progenitor cells. However, cohesin silencing in vivo rapidly altered stem cells homeostasis and myelopoiesis. Likewise, we found widespread changes in chromatin accessibility and expression of genes involved in myelomonocytic maturation and differentiation. Finally, aged cohesin knockdown mice developed a clinical picture closely resembling myeloproliferative disorders/neoplasms (MPNs), including varying degrees of extramedullary hematopoiesis (myeloid metaplasia) and splenomegaly. Our results represent the first successful demonstration of a tumor suppressor function for the cohesin complex, while also confirming that cohesin mutations occur as an early event in leukemogenesis, facilitating the potential development of a myeloid malignancy.

Little is known on post-transcriptional regulation of stem cell maintenance and differentiation. Here we characterize the role of Ddb1, a component of the CUL4-DDB1 ligase complex. Ddb1 is highly expressed in hematopoietic stem cells and its deletion leads to abrogation of hematopoiesis, targeting specifically transiently amplifying progenitor subsets. Ddb1 deletion in non-dividing lymphocytes had no discernible phenotypes. Ddb1 silencing activated the p53 pathway and lead to apoptosis. The abrogation of hematopoietic progenitor cells can be partially rescued by simultaneous deletion of p53. Interestingly, depletion of DDB1 in embryonic stem cell (ESC) does not affect survival or cell cycle progression but leads to loss of pluripotency, suggesting distinct roles of DDB1 in adult and embryonic stem cells. Mass-spectrometry revealed distinct interactions between DDB1 and DCAFs, the substrate-recognizing components of the CUL4 complex between cell types. Our studies identify the CUL4-DDB1 complex as a novel post-translational regulator of stem maintenance and differentiation.