Misregulation of enhancer activity leads to various developmental disorders including thalassemia, Cornelia de Lange syndrome, and Kabuki syndrome. The major enhancer regulator Mll4 belongs to the family of the Su(var)3- 9, Enhancer-of-zeste, Trithorax (SET) domain containing histone methyltransferases named COMplex of Proteins ASsociated with Set1 (COMPASS), which places methylation marks at lysine 4 of histone H3 (H3K4). Mll4 is essential for mammalian development and its heterozygous loss-of-function mutations lead to various human diseases including Kabuki syndrome and cancer. I recently demonstrated that Mll4 is the major enzyme depositing mono-methylation at H3K4 (H3K4me1), an enhancer-decorating epigenetic mark whose function remains elusive. Moreover, I have defined catalytic activity dependent and independent functions of Mll4 in enhancer modulation and stem cell differentiation, and have unveiled an antagonism between enhancer-binding epigenetic machineries in transcriptional control that could underlie the developmental consequences of the heterozygous loss-of-function mutations of Mll4 in human disease. Based on these findings, I hypothesize that Mll4 cooperates with key transcription factors and epigenetic modifiers to modulate enhancer activity, chromatin structure, and transcriptional outputs during stem cell maintenance and differentiation. The studies proposed here aim to elucidate the molecular mechanisms of enhancer regulation, and to provide insights for developing novel therapies targeting diseases driven by the loss of function of epigenetic modifiers. Specifically, the outlined research will 1) elucidate how epigenetic marks impact enhancer functions and cell fate transition; 2) determine the role of higher order chromatin structure in stem cell maintenance and differentiation; 3) identify novel factors and pathways involved in modulating enhancer activity and determining cell fate. To achieve the long-term career goal of defining the epigenetic mechanisms underlying mammalian development and disease, I will acquire training in biochemistry, proteomics, bioinformatics, stem cell biology, and genome-wide screening during the mentored phase of this application. Moreover, I will participate in grant writing workshops and career development courses to strengthen my skills in writing and leadership. With the acquired training, I will be well- prepared for the task of delineating the machineries and mechanisms in modulating gene expression, chromatin structure, and cell fate determination in the independent R00 phase. In summary, the K99/R00 award, together with the experiences that I have garnered as a graduate student and postdoc, the guidance from the mentors and collaborators, and the superb research environment at Northwestern University's Feinberg School of Medicine, will ensure a successful transition for me to continue my independent scientific career in the field of stem cell biology and epigenetics.

Public Health Relevance

Mutations in Mll4 lead to Kabuki syndrome and multiple types of cancer, but how Mll4 impacts development and human diseases is unknown. Here I propose to study the enhancer regulatory mechanisms by which Mll4 controls stem cell differentiation and early mammalian development, an important process underlying pathogenesis. Accomplishment of the proposed studies will provide novel drug targets for clinical applications to treat diseases driven by enhancer malfunction.

National Institute of Health (NIH)
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Career Transition Award (K99)
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National Institute of Child Health and Human Development Initial Review Group (CHHD)
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Mukhopadhyay, Mahua
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Northwestern University at Chicago
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United States
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