Bromodomain factor 5 is an essential regulator of transcription in Leishmania

Abstract

Leishmania are unicellular parasites that cause human and animal diseases. Like other kinetoplastids, they possess large transcriptional start regions (TSRs) which are defined by histone variants and histone lysine acetylation. Cellular interpretation of these chromatin marks is not well understood. Eight bromodomain factors, the reader modules for acetyl-lysine, are found across Leishmania genomes. Using L. mexicana, Cas9-driven gene deletions indicate that BDF1–5 are essential for promastigotes. Dimerisable, split Cre recombinase (DiCre)-inducible gene deletion of BDF5 show it is essential for both promastigotes and murine infection. ChIP-seq identifies BDF5 as enriched at TSRs. XL-BioID proximity proteomics shows the BDF5 landscape is enriched for BDFs, HAT2, proteins involved in transcriptional activity, and RNA processing; revealing a Conserved Regulators of Kinetoplastid Transcription (CRKT) Complex. Inducible deletion of BDF5 causes global reduction in RNA polymerase II transcription. Our results indicate the requirement of Leishmania to interpret histone acetylation marks through the bromodomain-enriched CRKT complex for normal gene expression and cellular viability.

Deciphering divergent trypanosomatid nuclear complexes by analyzing interactomic datasets with AlphaFold2 and genetic approaches

“…One of them is Tb927.9.13320, which possesses an FHA ( f ork h ead a ssociated) domain, a module involved in phospho-threonine recognition 30 . This protein network was proposed to be forming a complex only found in trypanosomatids that would act as a central regulator of transcription and RNA processing 15 . No MRGBPs were identified in this interaction network either.…”

“…They performed Co-IP assays of the 7 BDFs recognizable in the T. brucei genome, 9 proteins with acetyl transferase domains and 7 proteins identified as potential HDACs, among others. Another approach to this problem was that of Jones et al , who used a proximity labelling methodology to generate an interactomic dataset of BDF5 from L. mexicana ( Lmx BDF5), followed by Co-IP to validate some of the proximity hits as interactions, proposing a novel Conserved Regulator of Kinetoplastid Transcription (CRKT) complex 18 . Also, Vellmer et al 19 , while trying to unveil the identity and composition of a novel SNF2 complex involved in H2A.Z deposition, derived in the conclusion that some of the proteins they identified were forming two different HAT complexes, involving Tb HAT1 and Tb HAT2.…”

CLK1/CLK2-driven signalling at the Leishmania kinetochore is captured by spatially referenced proximity phosphoproteomics

“…A key component of our spatially referenced proximity phosphoproteomics approach was the use of BDF5 as a spatial reference 10 . BDF5 localises to chromatin, but to different regions than the kinetochore 19 . It, therefore, provides a compartment-matched set of background ‘bystander’ proteins and phosphosites that are captured by the workflow, but are not proximal to the kinetochore (Fig.…”

Chemical inhibition of bromodomain proteins in insect stage African trypanosomes perturbs silencing of the Variant Surface Glycoprotein repertoire and results in widespread changes in the transcriptome

“…In parasites, bromodomain inhibitors have been shown to bind to bromodomain proteins in T. brucei, T. cruzi, P. falciparum, and L. dovani 23,27,[41][42][43][44] . Inhibition of bromodomain proteins in parasites has been shown to affect differentiation processes in multiple parasite systems 23,45,46 , and therapeutic strategies targeting chromatin interacting proteins have also been proposed and/or demonstrated for trypanosomiasis 23,30 , Chagas disease [47][48][49] , schistosomiasis 50,51 , toxoplasmosis 52,53 , leishmaniasis 54 , and malaria 55,56 .…”