1) We have defined that p300 plays a central role the control of primary genetic response in human leukemic cells. 2) We have made the first description of the process of dynamic bookmarking of endogenous genes by the RNA polymerase II complex. We hypothesize that this complex conditionally marks gene for subsequent challenge by differential stimuli and defines a mechanism for molecular memory that may have a role physiological responses as diverse as drug resistance, drug addiction, immunological memory and cognitive memory. 3) By quantitative chromatin immunoprecipitation and genome-wide location analysis we found that p300 plays a role in the dynamic bookmarking of mitotic chromatin in manner that facilitates the transfer of molecular memory of formed transcriptional complexes at specific genes in the parental cells to progeny cells. Depletion of p300 results in disrupted formation of these complexes and reduced histone acetylation at specific genes of parental cell progeny. These findings implicate a major role for p300 in the propagation of epigenetic information during cell division in lymphoid derived and leukemia/lymphoma cells. 4.) Genome-wide analysis of the assembly of p300 in lymphoid leukemia cells reveals dramatic transition in the formation of p300-containing complexes as cell progress through the cell cycle. 5.) Gene replacement studies in human colon carcinoma cell lines deficient in p300 indicate a role for p300 in promoting resistance to mitotic inhibitors that interfere with mitotic spindle and possible other chemotherapeutic agents. 6.) We have found that there are high levels of p300-containing pick complexes in that are pre-load or bookmarked in human memory CD4 and CD8 T-cells in comparison to human nave CD4 and CD8 cells. 7.) We have found that p300 forms a complex with the Eleven-nineteen Lysine rich Leukemia protein (ELL) and the components of the positive transcriptional elongation factor (P-TEFb, composed of CDK9 + Cyclin T) through protein-protein interactions that are increased by mitogen stimulation. 8.) Extended studies that in addition to the co-assembly of p300, ELL and pTEFb complexes at the promoters of rapidly induced genes, pre-assembled complexes of p300/ELL/pTEFb can be detected specifically in the nucleus, whereas they remain disassembled in the cytoplasm. Mitogen stimulation primary human cells and leukemic cells cultures results in dramatic shift in the free and active fractions of these factors suggesting a major mode through which gene expression is regulated by controlling the availability of elongation factors components. 8) In genome-wide location analysis we have found that the elongation factor eleven-nineteen lysine rich leukemia protein (ELL) assembles in the coding regions of numerous genes where it travels with the transcriptionally engaged and elongating RNA polymerase. By employing fluorescence recovery after photo-bleaching we show that in vivo association of ELL with pol II leads to an approximate 10 fold increase in the transcriptional elongation efficiency of pol II. 9.) We have found that the C-terminal region of ELL is responsible for its interaction with the P-TEFb complex. This will allow future structure function correlation. 10.)We have successfully developed an antibody the recognizes ELL by immunoblot, immunfluorescence, immune-peroxidase staining in human tissue sections, by ELISA and by chromatin immune-precipitation. Using this antibody in human tissues sections we find that ELL is highly enriched in proliferating human lymphoid tissue, and shows strikingly high expression in the nuclei of placental syncytial-trophoblasts. 11.) We have found that pharmacological targeting of transcriptional elongation can synergized to enhance the effectiveness of ant-leukemia chemotherapy using redox reactive thalidomide analogs. 12.) By live cell imaging we demonstrate the ELL forms dynamic transcriptional complexes with distinct complexes during transcriptional initiation and transcriptional elongation. 13.) ELL is require for both pause site entry and pausing release of RNA polymerase II. 14.) ELL containing complexes are targeted for regulation by both leukemogenic MLL fusion protein and the HTLV-1 Tax oncoprotein.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC010846-07
Application #
8763249
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
7
Fiscal Year
2013
Total Cost
$303,919
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code