Efrat Shema's Lab

Despite advances in deciphering chromatin structure and dynamics in recent years, mapping the sequence position and combinatorial modifications of individual nucleosomes, the building blocks of chromatin, has yet to be achieved. In this work, we develop SM-NucSeq: a technology that combines single-molecule immunoaffinity detection with single-molecule real-time (SMRT) sequencing in zero-mode waveguides (ZMWs), which are sub-wavelength well-like structures that confine the depth of the excitation beam to the nanometer scale. We show that SM-NucSeq can detect histone modifications on intact nucleosomes and identify their underlying DNA sequences. We leverage the ZMW chips to load nucleosome/polymerase complexes and detect co-occurring histone modifications with fluorescent antibodies, validating each step on a chromatically resolved micromirror TIRF microscope. Finally, the application of SM-NucSeq reveals that DNA synthesis rates are reduced in nucleosomes compared to the double-stranded DNA control sample and that DNA synthesis rate profiles are histone-modification-dependent.

Breast cancer is the leading cause of death in women under 50. The majority of breast cancers are estrogen receptor α-positive (ER+) and are commonly treated with hormonal therapies such as tamoxifen that inhibit ER activity. The TP53 tumor suppressor gene, encoding the p53 protein, is the most frequently mutated gene in breast cancer, and TP53 mutations are associated with diminished tamoxifen response and worse prognosis for breast cancer patients. Here, we report that in breast cancer cells p53 and ER cooperate to regulate the transcription of a set of genes encoding chromatin modifiers. The net result is a global increase in H3K4me3 and decrease in H3K9me3 chromatin marks. The resultant \u201copen\u201d chromatin is associated with increased transcription of luminal cell identity genes and enhanced tamoxifen sensitivity. Conversely, diminished p53 control of these chromatin modulators is associated with the evolution of tamoxifen resistance and cancer stem cell properties.

Diffuse large B cell lymphomas and follicular lymphomas show recurrent mutations in epigenetic regulators; among these are loss-of-function mutations in KMT2D and gain-of-function mutations in EZH2. To systematically explore the effects of these mutations on the wiring of the epigenetic network, we applied a single-cell approach to probe a wide array of histone modifications. We show that mutant-EZH2 elicits extensive effects on the epigenome of lymphomas, beyond alterations to H3K27 methylations, and is epistatic over KMT2D mutations. Utilizing the single-cell data, we present computational methods to measure epigenetic heterogeneity. We identify an unexpected characteristic of mutant-EZH2, but not KMT2D, in increasing heterogeneity, shedding light on a novel oncogenic mechanism mediated by this mutation. Finally, we present tools to reconstruct known interactions within the epigenetic network, as well as reveal potential novel cross talk between various modifications, supported by functional perturbations. Our work highlights novel roles for mutantEZH2 in lymphomagenesis and establishes new concepts for measuring epigenetic heterogeneity and intra-chromatin connectivity in cancer cells.