Molecular regulation of BRD3 in forward programming of Megakaryocytes

Platelets are one of the most abundant cell type in the blood, and they play a crucial role in the process of homeostasis. Platelet traits, such as platelet count (PLT) and mean platelet volume (MPV) are highly heritable and stable within individuals, but the molecular mechanisms controlling these traits are poorly understood. Genome-wide association studies (GWAS) have identified BRD3 as a regulator of platelet traits. BRD3, along with BRD2, BRD4 and BRDT, is classified within the bromodomain and extra terminal (BET) family, specialised in recognising and binding to acetylated lysine residues. In this project, I studied the functions of BRD3 during megakaryopoiesis, the process that leads to platelet formation. Because platelets do not have nucleus, megakaryocytes are the best model to study chromatin interactors associated with platelet traits. I used CRISPR/Cas9 to generate a BRD3 KO iPSc model. These cells were capable to differentiate into MKs, using a forward programming protocol, demonstrating that BRD3 is not essential for MK differentiation. I found that a subset of genes was differentially expressed in the absence of BRD3, despite genome-wide chromatin accessibility and H3K27ac signatures remaining unaltered. In order to investigate whether there was a compensatory effect among BET proteins, I designed BRD2 and BRD4 KO iPSCs, as well as combinations of BET KOs. BRD2 KO generated MK progenitors, indicating that the protein is also dispensable in MK generation. Interestingly, using an inhibitor that recognises all BET proteins, MK progenitor differentiation was impaired, but not late megakaryopoiesis, suggesting that some BET proteins might play a critical role in early MK differentiation. Overall, these results indicate that BRD2 and BRD3 are dispensable in megakaryocytes differentiation, and probably, that BRD4 might be essential due to its role in mesoderm differentiation. Together, this work starts to unveil the requirement of BET during megakaryopoiesis.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2019

Author dc:creator

• Rosa, Maria Isabel

Advisor dc:contributor.advisor

• Frontini, Mattia

• Megakaryopoiesis
• bromodomains
• BRD3

Licence

• https://www.repository.cam.ac.uk/bitstreams/bcb9bc11-83c9-400c-96ac-a9b2aa94ecba/download
• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

en

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.39739

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/292580