NLP is a novel transcription regulator involved in VSG expression site control in Trypanosoma brucei

Abstract

Trypanosoma brucei mono-allelically expresses one of approximately 1500 variant surface glycoprotein (VSG) genes while multiplying in the mammalian bloodstream. The active VSG is transcribed by RNA polymerase I in one of approximately 15 telomeric VSG expression sites (ESs). T. brucei is unusual in controlling gene expression predominantly post-transcriptionally, and how ESs are mono-allelically controlled remains a mystery. Here we identify a novel transcription regulator, which resembles a nucleoplasmin-like protein (NLP) with an AT-hook motif. NLP is key for ES control in bloodstream form T. brucei, as NLP knockdown results in 45- to 65-fold derepression of the silent VSG221 ES. NLP is also involved in repression of transcription in the inactive VSG Basic Copy arrays, minichromosomes and procyclin loci. NLP is shown to be enriched on the 177- and 50-bp simple sequence repeats, the non-transcribed regions around rDNA and procyclin, and both active and silent ESs. Blocking NLP synthesis leads to downregulation of the active ES, indicating that NLP plays a role in regulating appropriate levels of transcription of ESs in both their active and silent state. Discovery of the unusual transcription regulator NLP provides new insight into the factors that are critical for ES control.

Transcription is initiated on silent variant surface glycoprotein expression sites despite monoallelic expression in Trypanosoma brucei

“…The precise underlying molecular mechanism is still not understood. Although several molecular players have been shown to be involved (8,(11)(12)(13)(14)(15)(16)(17)(18)(19)(20), depletion of any of these factors had only a moderate effect on the repression of silent VSG genes. Therefore, either a master regulator is still to be discovered or, alternatively, the process is controlled by multiple collaborative factors ensuring a cumulative tight lock.…”

“…The mechanism controlling the selective activity of a single ES at a time is still unknown, although several factors have been found to be involved, such as differential chromatin/histone acetylation and methylation between active and inactive ESs (6-8); nucleosome depletion in the active ES (9,10); and the influence of various negative transcription modulators such as a histone methylase, a high-mobility group protein, histone H1, NLP (a nucleoplasm-like protein), a FACT subunit (a chromatin remodeler), a SWI/SNF (a chromatin remodeling factor), ORC1 and MCM-BP (proteins involved in the initiation of DNA replication), RAP1 (a telomere-binding protein), histone chaperones, or a lamin-like protein (8,(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21). However, no master ES regulator has been identified so far.…”

“…Furthermore, the level at which the control takes place is unknown: Whether the transcription control takes place at the initiation or elongation level is debated. Data reporting low levels of transcripts from silent ESs upon RNAi-mediated knockdown of various factors (8,(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21) cannot resolve this issue, because the data are compatible with both models. Previous experiments pointed to mixed conclusions.…”

The VEXing problem of monoallelic expression in the African trypanosome

“…Depletion of histone H3, H1, or H3.V causes increased transcription at inactive BES promoters (8,9) or at VSGs themselves (10,11). Histone deposition factors and nucleosome remodelers have also been shown to be important for maintaining silencing at these sites (9,12). Interfering with proteins that read, write, or erase modifications on the histone tails can disrupt silencing of telomeric reporter genes or inactive BESs, depending on the factor (9,(13)(14)(15)(16)(17).…”

“…If one imagines VEX1 as a gatekeeper for entry to an ESBassociated VSG transcription "center," then loss of VEX1 would allow a "free-for-all" entry of additional BESs within the ESBassociated center and access to the transcription factors housed within it [Pol I, class I transcription factor A (CITFA), or nucleoplasminlike protein (NLP) (12,20), and possibly others], resulting in increased transcription of VSGs at the newly associated BESs. Conversely, overabundance of VEX1 would establish additional gated centers for VSG expression (which might not necessarily coincide with the ESB).…”