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The direct transcriptional regulation of cell cycle genes by cell identity factors during myeloid differentiation

Award Number: R15HL172092
ORGANIZATION: NATIONAL HEART, LUNG, & BLOOD INSTITUTE
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 09/01/2024
PERIOD OF PERFORMANCE END DATE: 08/31/2027

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The direct transcriptional regulation of cell cycle genes by cell identity factors during myeloid differentiation - Summary During blood development, most stem cells are quiescent, enter the cell cycle infrequently to amplify as short-term hematopoietic stem cells and lineage-restricted progenitors and then exit the cell cycle in order to permanently differentiate into mature and functional cell types. Cell cycle exit is relevant to cancer since leukemic stem cells (LSCs) undergo a similar process of expansion and exit during tumorigenesis. This project seeks to uncover the mechanisms by which cell cycle exit is achieved during terminal myeloid differentiation. While transcription factors (TFs) that specify myeloid cell identity, PU.1 and C/EBPα/β/δ, are known to inhibit the cell cycle, whether they directly regulate the expression of canonical cell cycle genes acting at the G1/S transition is not known. This project will investigate the hypothesis that PU.1 and C/EBP-family TFs bind to distal enhancers of cyclin- dependent kinase (CDK) inhibitors (CDKIs) and Cyclin D2 to upregulate or downregulate them respectively in a cell-identity speciﬁc manner. Aim 1 will test whether the identiﬁed CDKIs and Ccnd2 cause cell cycle exit in an in vitro model of myeloid differentiation. Gain of function experiments for the CDKIs Cdkn1a, Cdkn1b, Cdkn2c, and Cdkn2d and loss of function experiments for Ccnd2 will be performed and the proportion of cells exiting the cell cycle will be measured. How genome-wide gene expression and DNA accessibility changes with cell cycle will be determined by synchronizing cells and performing RNA-Seq and ATAC-Seq time course experiments. Cells that have exited during differentiation will be isolated using dye dilution techniques and their transcriptome and DNA accessibility will be compared to those of cycling cells to uncover new candidates for mediating cell cycle exit. Aim 2 is to determine how cell cycle genes are regulated by PU.1- and C/EBP-bound enhancers at the resolution of TF binding sites. A set of enhancers of the CDKIs and Ccnd2 has been identiﬁed using high-resolution DNA accessibility proﬁling (ATAC-Seq). The occupancy of PU.1 and C/EBP-family TFs at their binding-sites in these enhancers will be conﬁrmed using CUT&RUN. Putative enhancers will be tested using reporter genes integrated in a site-speciﬁc manner with CRISPR/Cas9 into the ROSA26 locus. Predicted binding sites will be validated using site-directed mutagenesis of the reporter locus. Whether PU.1 and/or C/EBP-family TFs binding to the candidate enhancers regulate their endogenous targets and cell cycle exit will be checked by mutating the validated binding sites in the endogenous locus with CRISPR/Cas9 homology directed repair (HDR).


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2024 ( Subtotal = $423,000 )
2024	2024	UNIVERSITY OF NORTH DAKOTA	264 CENTENNIAL DR STOP 8371	GRAND FORKS	ND	58202	GRAND FORKS	USA	Blood Diseases and Resources Research	000	1	8/12/2024	NEW	$423,000
														Subtotal = $423,000

Grand Total All Awards = $423,000

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The direct transcriptional regulation of cell cycle genes by cell identity factors during myeloid differentiation

Award Number: R15HL172092

ORGANIZATION: NATIONAL HEART, LUNG, & BLOOD INSTITUTE

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

PERIOD OF PERFORMANCE START DATE: 09/01/2024

PERIOD OF PERFORMANCE END DATE: 08/31/2027

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