The Putative Bromodomain Protein PfBDP7 of the Human Malaria Parasite Plasmodium Falciparum Cooperates With PfBDP1 in the Silencing of Variant Surface Antigen Expression

Abstract

Epigenetic regulation is a critical mechanism in controlling virulence, differentiation, and survival of the human malaria parasite Plasmodium (P.) falciparum. Bromodomain proteins contribute to this process by binding to acetylated lysine residues of histones and thereby targeting the gene regulatory machinery to gene promoters. A protein complex containing the P. falciparum bromodomain proteins (PfBDP) 1 and PfBDP2 (BDP1/BDP2 core complex) was previously shown to play an essential role for the correct transcription of invasion related genes. Here, we performed a functional characterization of a third component of this complex, which we dubbed PfBDP7, because structural modelling predicted a typical bromodomain fold. We confirmed that PfBDP7 is a nuclear protein that interacts with PfBDP1 at invasion gene promoters in mature schizont stage parasites and contributes to their transcription. Although partial depletion of PfBDP7 showed no significant effect on parasite viability, conditional knock down of either PfBDP7 or PfBDP1 resulted in the de-repression of variant surface antigens (VSA), which are important pathogenicity factors. This de-repression was evident both on mRNA and protein level. To understand the underlying mechanism, we mapped the genome wide binding sites of PfBDP7 by ChIPseq and showed that in early schizonts, PfBDP7 and PfBDP1 are commonly enriched in heterochromatic regions across the gene body of all VSA families, including genes coding for PfEMP1, RIFIN, STEVOR, and PfMC-2TM. This suggests that PfBDP7 and PfBDP1 contribute to the silencing of VSAs by associating with heterochromatin. In conclusion, we identified PfBDP7 as a chromatin binding protein that is a constitutive part of the P. falciparum BDP1/BDP2 core complex and established PfBDP1 and PfBDP7 as novel players in the silencing of heterochromatin regulated virulence gene families of the malaria parasite P. falciparum.

Structural insights into acetylated histone ligand recognition by the BDP1 bromodomain of Plasmodium falciparum

“…Our study establishes an additional activity of the PfBDP1-BRD in recognizing histone H4 and H2B.Z acetylation marks, whose functional significance with respect to the regulation of gene expression in P. falciparum has yet to be explored. The PfBDP1 protein has also been shown to form a multi-subunit complex with PfBDP2 and/or PfBDP7 [24, 27, 43]. In addition to the C-terminal bromodomain, the full-length PfBDP1 protein contains 7 ankyrin repeats located near the N-terminus, which may function as a protein-protein interaction domain.…”

“…The P. falciparum genome also encodes for ten bromodomain-containing proteins namely, Bromodomain Proteins 1-7 (PfBDP1, PfBDP2, PfBDP3, PfBDP4, PfBDP5, PfBDP6, PfBDP7, PfBDP8, GCN5 (histone acetyltransferase GCN5), and SET1 (SET domain protein 1)), which likely recognize acetylated lysine modifications. Recent studies on PfBDP1 have shown that it is a multi-domain protein with a predicted ankyrin repeat in the N-terminal region, and a bromodomain (BRD) near the C-terminus [16,24,[27][28][29]. PfBDP1 is thought to play an important role in the activation of invasion-related genes in the asexual stage as its knockdown drastically affects the replication of P. falciparum in red blood cells.…”

“…In addition, histone peptide pull-down assays also indicate that PfBDP1 can interact with acetyllysine modifications on histone H4, and the histone variants H2B.Z and H2B.A [22]. Interestingly, PfBDP1 appears to form a core complex with two other bromodomain-containing proteins, PfBDP2 and PfBDP7, which were also enriched with acetylated histones in the pull-down assays [22, 24, 27]. PfBDP7 is an essential gene in P. falciparum that was recently shown to co-localize with the genome-wide binding sites of PfBDP1.…”

“…Both proteins are commonly found at the promoters of invasion genes, however, PfBDP1 and PfBDP7 are also enriched in heterochromatin at genes encoding the variant surface antigens, which contribute to parasite infection. Thus, they are also thought to function as an important regulatory complex for silencing the expression of variant surface antigen [27]. Similarly to the SAGA and RSC chromatin remodeling complexes, which contain multiple bromodomain proteins, PfBDP7 has been shown to interact with PfBDP1 and PfBDP2, likely allowing the complex to recognize a specific subset of acetylation marks on histone proteins [22, 24, 27].…”

“…Thus, they are also thought to function as an important regulatory complex for silencing the expression of variant surface antigen [27]. Similarly to the SAGA and RSC chromatin remodeling complexes, which contain multiple bromodomain proteins, PfBDP7 has been shown to interact with PfBDP1 and PfBDP2, likely allowing the complex to recognize a specific subset of acetylation marks on histone proteins [22, 24, 27].…”