Abraham S. Weintraub

Convergence of developmental and oncogenic signaling pathways at transcriptional super-enhancers

Super-enhancers and stretch enhancers (SEs) drive expression of genes that play prominent roles in normal and disease cells, but the functional importance of these clustered enhancer elements is poorly understood, so it is not clear why genes key to cell identity have evolved regulation by such elements. Here, we show that SEs consist of functional constituent units that concentrate multiple developmental signaling pathways at key pluripotency genes in embryonic stem cells and confer enhanced responsiveness to signaling of their associated genes. Cancer cells frequently acquire SEs at genes that promote tumorigenesis, and we show that these genes are especially sensitive to perturbation of oncogenic signaling pathways. Super-enhancers thus provide a platform for signaling pathways to regulate genes that control cell identity during development and tumorigenesis.

YY1 Is a Structural Regulator of Enhancer-Promoter Loops

There is considerable evidence that chromosome structure plays important roles in gene control, but we have limited understanding of the proteins that contribute to structural interactions between gene promoters and their enhancer elements. Large DNA loops that encompass genes and their regulatory elements depend on CTCF-CTCF interactions, but most enhancer-promoter interactions do not employ this structural protein. Here, we show that the ubiquitously expressed transcription factor Yin Yang 1 (YY1) contributes to enhancer-promoter structural interactions in a manner analogous to DNA interactions mediated by CTCF. YY1 binds to active enhancers and promoter-proximal elements and forms dimers that facilitate the interaction of these DNA elements. Deletion of YY1 binding sites or depletion of YY1 protein disrupts enhancer-promoter looping and gene expression. We propose that YY1-mediated enhancer-promoter interactions are a general feature of mammalian gene control.

APOBEC signature mutation generates an oncogenic enhancer that drives LMO1 expression in T-ALL

Oncogenic driver mutations are those that provide a proliferative or survival advantage to neoplastic cells, resulting in clonal selection. Although most cancer-causing mutations have been detected in the protein-coding regions of the cancer genome; driver mutations have recently also been discovered within noncoding genomic sequences. Thus, a current challenge is to gain precise understanding of how these unique genomic elements function in cancer pathogenesis, while clarifying mechanisms of gene regulation and identifying new targets for therapeutic intervention. Here we report a C-to-T single nucleotide transition that occurs as a somatic mutation in noncoding sequences 4 kb upstream of the transcriptional start site of the LMO1 oncogene in primary samples from patients with T-cell acute lymphoblastic leukaemia. This single nucleotide alteration conforms to an APOBEC-like cytidine deaminase mutational signature, and generates a new binding site for the MYB transcription factor, leading to the formation of an aberrant transcriptional enhancer complex that drives high levels of expression of the LMO1 oncogene. Since APOBEC-signature mutations are common in a broad spectrum of human cancers, we suggest that noncoding nucleotide transitions such as the one described here may activate potent oncogenic enhancers not only in T-lymphoid cells but in other cell lineages as well.

Transcriptional Dysregulation of MYC Reveals Common Enhancer-Docking Mechanism

SUMMARY Transcriptional dysregulation of the MYC oncogene is among the most frequent events in aggressive tumor cells, and this is generally accomplished by acquisition of a super-enhancer somewhere within the 2.8 Mb TAD where MYC resides. We find that these diverse cancer-specific super-enhancers, differing in size and location, interact with the MYC gene through a common and conserved CTCF binding site located 2 kb upstream of the MYC promoter. Genetic perturbation of this enhancer-docking site in tumor cells reduces CTCF binding, super-enhancer interaction, MYC gene expression, and cell proliferation. CTCF binding is highly sensitive to DNA methylation, and this enhancer-docking site, which is hypomethylated in diverse cancers, can be inactivated through epigenetic editing with dCas9-DNMT. Similar enhancer-docking sites occur at other genes, including genes with prominent roles in multiple cancers, suggesting a mechanism by which tumor cell oncogenes can generally hijack enhancers. These results provide insights into mechanisms that allow a single target gene to be regulated by diverse enhancer elements in different cell types.

Corrigendum: Small genomic insertions form enhancers that misregulate oncogenes

This corrects the article DOI: 10.1038/ncomms14385.

PHF6 regulates phenotypic plasticity through chromatin organization within lineage-specific genes

Developmental and lineage plasticity have been observed in numerous malignancies and have been correlated with tumor progression and drug resistance. However, little is known about the molecular mechanisms that enable such plasticity to occur. Here, we describe the function of the plant homeodomain finger protein 6 (PHF6) in leukemia and define its role in regulating chromatin accessibility to lineage-specific transcription factors. We show that loss of Phf6 in B-cell leukemia results in systematic changes in gene expression via alteration of the chromatin landscape at the transcriptional start sites of B-cell- and T-cell-specific factors. Additionally, Phf6KO cells show significant down-regulation of genes involved in the development and function of normal B cells, show up-regulation of genes involved in T-cell signaling, and give rise to mixed-lineage lymphoma in vivo. Engagement of divergent transcriptional programs results in phenotypic plasticity that leads to altered disease presentation in vivo, tolerance of aberrant oncogenic signaling, and differential sensitivity to frontline and targeted therapies. These findings suggest that active maintenance of a precise chromatin landscape is essential for sustaining proper leukemia cell identity and that loss of a single factor (PHF6) can cause focal changes in chromatin accessibility and nucleosome positioning that render cells susceptible to lineage transition.

Abstract 2004: Nucleation of transcriptional super-enhancers at tumor oncogenes by small insertions

Transcriptional super-enhancers drive expression of oncogenes in many cancers and are being targeted with novel transcriptional and epigenetic therapeutics[1,2,3,4]. Super-enhancers are acquired by cancers through multiple mechanisms, including DNA translocation of an extant super-enhancer and focal amplification. We recently discovered a novel mechanism by which super-enhancers are nucleated in T cell acute lymphoblastic leukemias (T-ALLs)[5]. In this case, a small, monoallelic insertion creates a DNA binding site for a master transcription factor protein, which binds and recruits additional factors to nucleate the super-enhancer, which in turn drives high levels of the TAL1 oncogene. We describe here a method for unbiased identification of similar genomic insertions that nucleate potentially oncogenic regulatory elements in cancers. This approach uses data from genome-wide ChIP-Seq studies that map locations of enhancer-binding proteins to identify short DNA sequences missing from reference genomes. We have found and catalogued many additional genomic insertions in 80 additional cancers. An additional insertion has been functionally characterized and determined to be a bona fide enhancer-nucleating insertion that exists in patient genomes. I will describe new insights into the regulation of cancer that occur due to nucleation of novel regulatory elements. [1] Hnisz, Abraham, Lee, et al., Cell 2013 [2] Loven, Hoke, Lin, et al., Cell 2013 [3] Kwiatkowski, et al., Nature, 2014 [4] Wang, et al., Cell, 2015 [5] Mansour et al., Science 2014 Citation Format: Brian J. Abraham, Denes Hnisz, Abraham S. Weintraub, Nicholas Kwiatkowski, Charles H. Li, Sunniyat Rahman, Zhaodong Li, Tong Ihn Lee, A Thomas Look, Marc Mansour, Richard A. Young. Nucleation of transcriptional super-enhancers at tumor oncogenes by small insertions. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2004.

Abstract A44: The role of PHF6 in maintaining pre-B cell commitment in B-cell acute lymphoblastic leukemia

Loss of function mutations in the plant homeodomain factor 6 (PHF6) are responsible for the Borjeson–Forssman–Lehmann syndrome, a familial X-linked intellectual disability disorder, and are observed in approximately 20% of adult T-cell acute lymphoblastic leukemias (T-ALLs) and 3% of adult acute myeloid leukemias (AMLs). However, mutations in B-cell lineage malignancies are notably absent. Interestingly, our recent work has uncovered PHF6 as a positive growth regulator in B-cell acute lymphoblastic leukemia (B-ALL) through a genome-scale in vivo loss-of-function screen. To identify the molecular mechanism by which PHF6 acts to promote B-ALL growth in vivo, we utilized CRISPR-Cas9 to delete Phf6 in murine B-ALL cells. Transplantation of Phf6 knockout cells (Phf6KO) into immunocompetent syngeneic recipients significantly extends disease latency and survival, therefore validating PHF6 as a bona fide positive growth regulator of B-ALL in vivo. Strikingly, these mice develop lymphoma-like disease with complete penetrance, characterized by significantly enlarged lymph nodes, decreased disease burden in the spleen and increased expression of the canonical T-cell marker CD4, suggesting that Phf6KO B-ALL cells transdifferentiated to cells resembling those of the T-cell lineage. To dissect the mechanism by which PHF6 regulates this lineage decision, we carried out a combination of RNA sequencing and chromatin immunoprecipitation (ChIP) analyses in Phf6WT and Phf6KO cells. RNA sequencing analysis revealed many differentially expressed genes in Phf6KO B-ALL cells , including gene sets involved in pathways important for B-cell development. ChIP-sequencing analysis of PHF6 and several histone marks (H3K27Ac, H3K27me3, H3K4me3) in Phf6WT B-ALL cells revealed that PHF6 and H3K27Ac signals co-localize close to the transcription start site of a significant proportion of differentially expressed genes. Transcription factor binding motif analysis revealed significant enrichment for several well-described master regulators of B-cell development, including PU.1, EGR-1, EBF-1, NF-κB and TCF3/TCF12. Notably, a number of these predicted transcription factors co-immunoprecipitated with PHF6 in Phf6WT B-ALL cells. These findings reveal an essential role for PHF6 in the maintenance of B-cell identity in B-ALL by activating, directly or indirectly, genes that are crucial for B-cell lineage commitment. Collectively, these results indicate that loss-of-function of PHF6 in B-ALL leads to transdifferentiation to the T-cell lineage, potentially explaining the apparent absence of PHF6 mutations in human B cell-lineage malignancies. Citation Format: Yadira M. Soto-Feliciano, Jordan ME Bartlebaugh, Yunpeng Liu, Francisco J. Sanchez-Rivera, Abraham S. Weintraub, Arjun Bhutkar, Tyler E. Jacks, Richard A. Young, Michael T. Hemann. The role of PHF6 in maintaining pre-B cell commitment in B-cell acute lymphoblastic leukemia. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr A44.

Abstract PR06: Nucleation of transcriptional super-enhancers at tumor oncogenes

Transcriptional super-enhancers drive expression of oncogenes in many cancers and are being targeted with novel transcriptional and epigenetic therapeutics (1,2,3,4). Super-enhancers are acquired in cancers through multiple mechanisms, including DNA translocation of an extant super-enhancer and focal amplification. We recently discovered a novel mechanism by which super-enhancers are nucleated in T cell acute lymphoblastic leukemias (T-ALLs) (5). In this case, a small, monoallelic insertion creates a DNA binding site for a master transcription factor protein, which binds and recruits additional factors to nucleate the super-enhancer, which in turn drives high levels of the TAL1 transcription factor. We describe here a method for unbiased identification of similar genomic insertions that nucleate potentially oncogenic regulatory elements in cancers. This approach uses data from genome-wide ChIP-Seq studies that map locations of enhancer-binding proteins to identify sequences missing from reference genomes. We have found many additional genomic insertions in many additional cancers. I will describe new insights into the regulation of cancer that occur due to nucleation of novel regulatory elements. 1 Hnisz, Abraham, Lee, et al., Cell 2013 2 Loven, Hoke, Lin, et al., Cell 2013 3 Kwiatkowski, et al., Nature, 2014 4 Chipumoro, et al., Cell, 2014 5 Mansour et al., Science 2014 Citation Format: Brian J. Abraham, Nicholas Kwiatkowski, Abraham S. Weintraub, Denes Hnisz, Nancy Hannett, Richard A. Young. Nucleation of transcriptional super-enhancers at tumor oncogenes. [abstract]. In: Proceedings of the AACR Special Conference on Computational and Systems Biology of Cancer; Feb 8-11 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 2):Abstract nr PR06.