Cell & Molecular Biology Graduate Group

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Jennifer Cohen, Sundaram Lab (matriculated 2014)

cohenI have always been fascinated with life's intricate shapes and the ways these forms impact function. My project focuses on the way an apical extracellular matrix (aECM) is organized and how it shapes a narrow tube. aECMsline most apical surfaces, including the outer surface of the skin, the inside of the lung, and the inside of blood vessels.However,aECMs are generally poorly characterized. aECMs often contain distinct layers whose structure and function is not well understood.Through forward genetic screening, we identified components of an aECM in the model organism,C.elegans. These components include a protein with a Zona Pellucida (ZP) domain-the same domain that polymerizes to form the Zona Pellucida, the oocyte's extracellular coat. As it turns out, ZP domains are pretty complex. Some may polymerize via as yet unknown mechanisms, and others may not polymerize at all! One part of my project has focused on understanding how our ZP domain protein functions– whether it is involved in polymerization,what part of the ZP domain is most important, whether it is cleaved and if cleavage is important, and what recruits the protein to its layer of the aECM. The second part of my project concerns a gene that came from a second forward genetic screen - this time, for mutations that bypass the requirement for this ZP domain protein. Through this screen, we identified loss-of-function mutations in a scavenger receptor gene. Scavenger receptors are transmembrane proteins that may transport cargoes to specific intracellular vesicles or pass hydrophobic molecules (like lipids) across membranes. This part of the project has the exciting possibility of uncovering connections between intracellular trafficking, lipid metabolism, and the aECM.

   

Yong Hoon Kim, Lazar Lab (matriculated 2012)

kimThe Lazar lab studies the transcriptional regulation of circadian rhythms and metabolism, with a specific focus on how nuclear hormone receptors and their co-regulators sense environmental cues to influence the epigenome. Circadian (circa “around” + dian “day”) rhythms are biological clocks that coordinate nearly all aspects of physiology to enable organisms on Earth to mount anticipatory and adaptive responses to environmental changes recurring every 24 hours. The molecular clock governing this process is orchestrated by transcription factors (TF) that function as activators and repressors in interlocking negative feedback loops. Throughout the genome, these TFs bind non-coding cis-regulatory elements called enhancers that often lie far from their target genes in the linear sequence, but physically loop to promoters to drive rhythmic gene transcription. One of the core clock TFs is the repressive nuclear receptor Rev-erb alpha, which controls a negative arm of the molecular clock by repressing transcription via recruitment of the epigenomic modulator Nuclear receptor Co-Repressor (NCoR) and Histone Deacetylase 3 (HDAC3) complex. My research focuses on understanding the circadian dynamics of enhancer-promoter loops and more specifically the role of Rev-erb alpha in regulating enhancer-promoter loops as a transcriptional repressor. Using mouse livers as an in vivo model and applying various genome-wide methods, we have demonstrated that circadian transcription is driven by rhythmic looping between enhancers and promoters, and that Rev-erb alpha represses transcription by opposing loop formation. At the molecular level, we uncovered that Rev-erb alpha opposes looping by deacetylating histones and evicting chromatin-associated factors that mediate looping. Our work highlights genome-wide plasticity in chromatin organization that occurs in a circadian manner as a component of normal mammalian physiology. Furthermore, it unveiled the previously unappreciated role of Rev-erb alpha in opposing enhancer-promoter loops, a mechanism which may be likely applicable to other transcriptional repressors whose function in controlling enhancer-promoter loops is not as well defined as for transcriptional activators.

 

Jeremy Grevet, Blobel Lab (matriculated 2011)

grevetMy work in the Blobel lab has focused on using CRISPR-based genetic screens to study hemoglobin switching. Increasing fetal hemoglobin (HbF) levels in adult red blood cells provides clinical benefit in patients with sickle cell disease (SCD) and some forms of β-thalassemia. While developing genome editing and gene replacement approaches promise long term HbF elevation in SCD patients, there remains a strong need for pharmacologic treatment of these diseases. Two transcription factors, BCL11A and LRF (ZBTB7A) and their co-regulators mediate most of fetal globin transcriptional silencing in adult erythroid cells. However, transcription factors are inherently challenging to inhibit with small molecules.

To identify potentially druggable HbF regulators, we carried out a CRISPR-Cas9 based genetic screen in a human erythroid cell line targeting protein kinases as they are inherently amenable to inhibition by small molecules. Most CRISPR-screens published to date have made use of sgRNAs targeting early exons to induce frameshift mutations. It has recently been shown that targeting sgRNAs to functional protein domains significantly improves screening efficiencies. We thus designed a sgRNA library targeting almost all annotated kinase-domains using a newly optimized sgRNA-scaffold that improves on-target editing activity. The screen uncovered the heme-regulated inhibitor HRI (also known as EIF2AK1) as an HbF repressor. HRI is an erythroid specific kinase known to regulate protein translation initiation.

HRI depletion increased the fraction of HbF+ cells and elevated fetal globin mRNA and protein levels. These effects were surprisingly specific as evidenced by whole proteome mass spectrometry and RNA-seq. Importantly, HRI depletion did not adversely affect cell growth or maturation. Similar results were obtained in primary CD34+ derived erythroid cells that were HRI depleted. HRI deficiency in erythroid cultures from patients with SCD reduced cell sickling, suggesting that biologically relevant HbF levels might be achieved by targeting HRI. Mechanistically, HRI loss strongly reduces BCL11A expression without impacting LRF production. BCL11A appears to be a major effector of HRI function since forced expression of BCL11A in HRI-depleted cells restores HbF repression to a significant degree. We are now further working up these findings mechanistically, and testing HRI depletion in combination with HbF inducing drugs.