Bromodomain factor 5 is an essential transcriptional regulator of the Leishmania genome

Abstract

Leishmania are unicellular parasites that cause human and animal disease. Alongside other organisms in kinetoplastida, they have evolved an unusual genome architecture that requires all RNA polymerase II transcribed genes to be expressed constitutively, with transcriptional start regions denoted by histone variants and histone lysine acetylation. However, the way these chromatin marks are interpreted by the cell is not understood. Seven predicted bromodomain factors (BDF1-7), the reader modules for acetyl-lysine, were identified across Leishmania genomes. Using L. mexicana as a model, Cas9-driven gene deletions indicate that BDF1-5 are essential for promastigote survival, whilst DiCre inducible gene deletion of the dual bromodomain factor BDF5 identified it to be essential for both promastigotes and amastigotes. ChIP-seq assessment of BDF5s genomic distribution revealed it as highly enriched at transcriptional start sites. Using an optimised proximity proteomic and phosphoproteomic technique, XL-BioID, we defined the BDF5-proximal environment to be enriched for other bromodomain factors, histone acetyltransferase 2, and proteins essential for transcriptional activity and RNA processing. Inducible deletion of BDF5, led to a disruption of pol II transcriptional activity and global defects in gene expression. Our results indicate the requirement of Leishmania to interpret histone acetylation marks for normal levels of gene expression and thus cellular viability.

Genomic Occupancy of the Bromodomain Protein Bdf3 Is Dynamic during Differentiation of African Trypanosomes from Bloodstream to Procyclic Forms

“…If an increase in acetylation levels and increased transcription are necessary during the transition from the quiescent stumpy form to the cycling procyclic form, this could result in the observed increase in Bdf3 occupancy following the differentiation cue. Bromodomain protein-mediated regulation of global Pol II transcript levels has been demonstrated in Leishmania ( 62 ). Transcriptome sequencing (RNA-seq) analyses are typically normalized in such a way as to obscure global increases or decreases in transcript levels ( 63 ).…”

Tag Thy Neighbour: Nanometre-Scale Insights Into Kinetoplastid Parasites With Proximity Dependent Biotinylation

“…Our group has recently completed two projects where we extended the use of proximity biotinylation to understanding fundamental aspects of chromatin biology in Leishmania (Geoghegan et al, 2021;Jones et al, 2021). We firstly discovered that we could increase the capture of proximal proteins by performing a limiting DSP cross-linking step after proximity biotinylation, an approach we termed XL-BioID (Geoghegan et al, 2021).…”

Essential bromodomain TcBDF2 as a drug target against Chagas disease

“…According to the TriTrypDB (http://tritrypdb.org/tritrypdb/), there are seven coding sequences for BD-containing proteins in trypanosomatids, named Tc BDF1 to Tc BDF7. An eighth putative bromodomain factor recently identified in Leishmania spp has an orthologue in T. cruzi 8 . Even though a few Tc BDFs have recognizable additional domains the architecture of these proteins seems to be simpler than those of BD-containing proteins from fungi and animals, and none contains histone acetyltransferase (HAT) domains, as occurs in at least one bromodomain-containing protein from almost all eukaryotic cells including many protists.…”

“…Even though a genome-wide RNA interference viability screen showed that Tb BDF2 was not essential, it was found essential in another assay 46 . Very recently, Jones et al showed that five BDFs, among them BDF2, are essential for Leishmania mexicana promastigotes 9 .…”

“…The final gene expression levels are regulated by several post-transcriptional mechanisms, which are not yet completely understood 5,6 . In parallel, transcription appears to be controlled by several proteins, histones and non-histones, which take part in protein complexes that regulate chromatin accessibility and are now starting to be identified [7][8][9] . Chromatin structure can be altered by posttranslational modifications in the N-terminal tails of histones, by the substitution of one or more histones by their variants, or by combinations of these events 10 .…”