Histone acetylations mark origins of polycistronic transcription in Leishmania major
Sean Thomas
2
Amanda Green
1
Nancy R Sturm
0
David A Campbell
0
Peter J Myler
1
3
4
0
Department of Microbiology, Immunology
,
and Molecular Genetics
,
University of California Los Angeles
,
609 E. Charles E Young Dr, Los Angeles, CA, 90095
,
USA
1
Seattle Biomedical Research Institute
,
307 Westlake Ave N. Ste 500, Seattle, WA 98109-5219
,
USA
2
Department of Genome Sciences, University of Washington
,
Seattle, WA 98195
,
USA
3
Department of Medical Education and Biomedical Informatics, University of Washington
,
Seattle, WA 98195
,
USA
4
Department of Global Health, University of Washington
,
Seattle, WA 98195
,
USA
Background: Many components of the RNA polymerase II transcription machinery have been identified in kinetoplastid protozoa, but they diverge substantially from other eukaryotes. Furthermore, protein-coding genes in these organisms lack individual transcriptional regulation, since they are transcribed as long polycistronic units. The transcription initiation sites are assumed to lie within the 'divergent strand-switch' regions at the junction between opposing polycistronic gene clusters. However, the mechanism by which Kinetoplastidae initiate transcription is unclear, and promoter sequences are undefined. Results: The chromosomal location of TATA-binding protein (TBP or TRF4), Small Nuclear Activating Protein complex (SNAP50), and H3 histones were assessed in Leishmania major using microarrays hybridized with DNA obtained through chromatin immunoprecipitation (ChIP-chip). The TBP and SNAP50 binding patterns were almost identical and high intensity peaks were associated with tRNAs and snRNAs. Only 184 peaks of acetylated H3 histone were found in the entire genome, with substantially higher intensity in rapidly-dividing cells than stationary-phase. The majority of the acetylated H3 peaks were found at divergent strand-switch regions, but some occurred at chromosome ends and within polycistronic gene clusters. Almost all these peaks were associated with lower intensity peaks of TBP/SNAP50 binding a few kilobases upstream, evidence that they represent transcription initiation sites. Conclusion: The first genome-wide maps of DNA-binding protein occupancy in a kinetoplastid organism suggest that H3 histones at the origins of polycistronic transcription of protein-coding genes are acetylated. Global regulation of transcription initiation may be achieved by modifying the acetylation state of these origins.
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Background
Kinetoplastid disease
Kinetoplastids are early-branching protists with unusual
mechanisms of gene expression. While some are harmless
free-living organisms, other members of this group infect
a range of plants and animals, causing significant human
disease in the form of African Sleeping Sickness
(Trypanosoma brucei), Chagas disease (Trypanosoma cruzi), and
leishmaniasis (Leishmania major), which kill
approximately 400,000 people per year. The parasites are
transmitted to their preferred hosts by different insect vectors
where they reside and replicate as host-adapted and
vector-adapted forms, respectively, with remarkably different
morphologies.
Leishmania are transmitted by the bite of a sand fly, where
they dwell in the mid-gut as promastigotes. The parasites
make their way to the salivary glands where they undergo
metacyclogenesis to a form infective to humans when the
sand fly feeds on the victim's blood. Once inside the host
bloodstream they are ingested by macrophages, where the
parasites can escape the host immune system and
transform into the amastigote form. Leishmaniasis symptoms
depend greatly on the infecting species of Leishmania and
present as one of three main types: a self-resolving
cutaneous form, a mucocutaneous form that destroys soft tissue
and cartilage in the face, and a more lethal visceral form
that infects the internal organs.
Regulation of gene expression in kinetoplastids
Kinetoplastids display peculiar molecular mechanisms,
especially when it comes to gene expression. In the
nucleus, functionally unrelated genes are transcribed
polycistronically [1] and are processed into individual
mature transcripts by trans-splicing, acquiring a 39-nt
mini-exon from the spliced leader (SL) RNA that is
attached to the 5' end of each individual messaged before
it can be translated. While bacteria use polycistronic
transcription as a method of co-regulating genes within an
operon, kinetoplastid genes are not typically arranged by
function [2], and it is thought that the steady-state levels
of proteins in kinetoplastid cells are determined
post-transcriptionally. The organization of genes on kinetoplastid
chromosomes also reflects this high degree of
polycistronic transcription; such that protein-coding genes on
chromosome 1 of L. major are arranged in only two long
gene clusters units, on opposite strands separated by a
'divergent strand-switch' region [3]. RNA polymerase
IImediated polycistronic transcription has been shown to
initiate within this strand-switch re (...truncated)