TINTIN, at the interface of chromatin, transcription elongation, and mRNA processing

Abstract

Recent work including high-resolution genome-wide analysis uncovered a new trimeric complex involved in transcription elongation, both as an integral part of the NuA4 histone acetyltransferase and as an independent functional entity. The complex is conserved in eukaryotes and is named TINTIN, for Trimer Independent of NuA4 for transcription Interactions with Nucleosomes. This point of view covers the current knowledge regarding TINTIN's function in modulating chromatin structure and influencing transcription elongation in eukaryotes. It also points to several physical and functional links to co-transcriptional processes, including interactions with the mRNA splicing machinery and the nuclear exosome.

NuA4/TIP60 for Nucleosome Transactions

Biogenesis of mRNA is vital for translating genetic information into protein. It is a multistep event involving transcription and mRNA processing in the nucleus followed by export of mRNA to the cytoplasm where protein synthesis takes place. All the steps in this process are coupled and tightly regulated to ensure that only properly processed mRNA is efficiently exported to cytoplasm.^1,2 Chromatin structure and dynamics plays a critical role in regulating transcription³ and also appears to be central to the process of linking transcription to mRNA processing steps.⁴

As with any other DNA related process, the different stages of transcription are profoundly affected by chromatin structure, which is specifically modulated so that transcriptional machinery can access DNA.³ Specific post-translational modifications of histones within chromatin play critical roles in the process. The majority of chromatin modifying enzymes can only act on chromatin when functioning as part of a multiprotein assembly. One essential and highly conserved chromatin modifying enzyme is the NuA4 (TIP60 in mammals)⁵ histone acetyltransferase complex. NuA4 has been implicated in the regulation of gene transcription through local recruitment by DNA bound transcription factors^6-9 or by histone marks and other regulators.^10-13 Notably NuA4 contains subunits, which are also present in other chromatin modifying enzymes. Of particular interest is chromodomain containing subunit Eaf3. It not only resides in NuA4, but is also a subunit of another complex with opposing activity, the Rpd3S histone deacetylase complex. While NuA4 is generally linked to transcriptional activation by its acetylation activity in “opening” promoter nucleosomes,^6,9 Rpd3S is associated with repressed chromatin structure on coding regions. Rpd3S deacetylates the nucleosomes and ensures proper refolding of chromatin and inhibition of spurious transcription from cryptic promoters within the coding region of genes.^14-16 Interestingly the chromodomain of Eaf3 interacts with H3K36me3 and recruits Rpd3S to the gene coding regions.^14-16 It remained puzzling for some time, why Eaf3 directs only Rpd3S to H3K36me3 over gene bodies, but not NuA4? Subsequently it was shown that combined action of PHD domain of Rco1 and chromodomain of Eaf3 specify recognition of the methyl H3K36 mark by the Rpd3S complex.¹⁷

Adding to the complexity, we recently provided evidence for the presence of Eaf3 in another small complex consisting of Eaf5/Eaf7/Eaf3.¹⁸ Tandem affinity purification followed by gel filtration clearly demonstrated that the Eaf5/7/3 trimer exists as a native complex outside of NuA4. We further showed that eaf5/7 mutant cells share similar phenotypes, in which some are shared with other components of NuA4 while others are specific to the trimer, further supporting the notion of an independent function. Due to its biochemical characteristics and molecular functions, this newly characterized sub-module is named as the TINTIN complex, for Trimer Independent of NuA4 involved in Transcription Interactions with Nucleosomes. The Eaf7 homolog in higher eukaryotes MRGBP interacts directly with MRG15, the human homolog of Eaf3. Gel filtration and a large-scale proteomic study indicate that the MRGBP-MRG15 complex also exists independently of the TIP60 complex.¹⁹ Results in our lab (K. Jacquet and J. Côté; in preparation) also demonstrate the distinct function of MRGBP-MRG15. Since there is no homolog of Eaf5 in human cells, MRGBP-MRG15 dimer is likely the functional homolog of the yeast TINTIN complex.

Our studies clearly demonstrate that Eaf5/7/3 exists as a separate entity as well as part of the larger NuA4 complex. While we showed that TINTIN tethers NuA4 through Eaf5, preliminary results indicate that the binding of TINTIN to NuA4 is mediated by its interaction with NuA4 scaffold subunit Eaf1 (AL. Steunou, J. Cote, unpublished data). However, it is still not known how this association to NuA4 is regulated. It is possible that the selective association/detachment of TINTIN to NuA4 is regulated through different cell cycle stages or in response to upstream signaling regulated by post-translational modifications (PTMs) on either protein. The identification of the TINTIN complex further highlights the major challenge of shared or common subunits in the study of chromatin modifying complexes. It is thus required to proceed with caution when selecting targets to investigate the specific roles of these multisubunit players.

TINTIN role in transcription elongation

Native chromatin structure is intrinsically incompatible with elongating RNAPII. During transcription elongation, chromatin structure is modulated in order to allow access to elongating RNAPII.^3,20 However, the proper refolding of chromatin after transcription by RNAPII is of utmost importance to the cell. Detrimental consequences that result from its deficiency include spurious transcription from cryptic promoters within the coding region of gene.^21,22 Histone chaperones, chromatin modifying enzymes and ATP dependent chromatin remodelers^23,24 are all key factors necessary for proper chromatin refolding.

Compared to NuA4, TINTIN complex is more highly enriched over coding regions relative to promoters in genomic mapping, suggesting the involvement in transcription elongation.¹⁸ Furthermore, we showed that the trimer travels with elongating Pol II over the gene bodies by utilizing a dual interaction surface with both H3K36me3 and phosphorylated RNAPII (Ser2). Both interactions are required for normal association on the body of transcribed genes, but Ser2-P on Pol II C-terminal domain (CTD) could be more important for targeting while H3K36me3 may be more linked to action on nucleosome dynamics, as is the case for Rpd3S.^25,26 This association to coding regions seems to destabilize nucleosomes, as trimer mutants suppress cryptic transcription detected in set2 deletion strains. Intriguingly, by interacting with histone chaperons, TINTIN prevents the incorporation of new histones and helps recycle/refold nucleosomes after the passage of pol II. Some of the critical players in chromatin refolding and histone exchange include histone chaperones Spt6, FACT, Asf1 and the chromatin remodelers Chd1 and Isw1.²⁴ It will be interesting to study whether there is crosstalk between TINTIN and these key players of chromatin refolding. If so, the underlying mechanisms would be pursued. Further analysis including mutant effects on cryptic transcription or the global transcriptome by RNA-seq would shed light on this pathway.

TINTIN and mRNA processing

It is well-established that mRNA processing occurs during transcription. Alternative splicing (AS) requires a vast array of proteins that catalyze splicing events. Both optimal RNA polymerase II elongation rate as well as the Pol II CTD play critical role in mRNA processing.^2,27-29 Chromatin structure and epigenetics also play important roles in co-transcriptional splicing.^4,30 One of the key events in the AS process is recognition of the splice site by proteins that either promote or suppress splicing. Polypyrimidine-tract binding protein (PTBP) is a splice junction-binding protein that suppresses splicing resulting in exon exclusion. PTB-dependent genes are enriched in H3K36me3. The high levels of H3K36me3 along the gene attract MRG15, which in turn interacts with PTB, recruiting it to the nascent RNA.⁴ Furthermore, the MRGBP-MRG15 dimer has now been linked to the splicing process in mammals linked to pausing of elongating polymerases carrying Ser2-P on their CTD.³¹ In yeast, TINTIN is associated with highly expressed genes. Interestingly, most of yeast intron-containing genes are highly expressed ribosomal protein (RP) genes. Moreover, TINTIN shows functional interaction with SWR1-C and Bdf1,^18,32,33 important regulators of alternative splicing.³⁴ It is likely that the trimer plays a similar role as MRGBP-MRG15 in splicing. Further study on the role of TINTIN in mRNA splicing will shed more light on this critical process in the context of chromatin.

TINTIN and mRNA quality control

Secondary mass-spectrometry hits in Eaf7-TAP purifications also include proteins associated with the exosome³⁵ (and our unpublished data). An important quality control for mRNA production is the degradation of aberrant transcripts by the exosome. The exosome is a highly conserved multiprotein complex with 3-5’ exonuclease activity.^36,37 Depletion of exosome subunits results in an increase in transcription read-through.^38,39 Interestingly we detected a small but reproducible increase of transcription read-through in eaf5/7 mutants using a reporter plasmid. Importantly, eaf7 and eaf5 mutants also show a negative genetic interaction with several components of the nuclear exosome.⁴⁰ Since mRNA biogenesis/processing events are tightly coupled, it is not surprising that eaf5/eaf7 also show negative interaction with the THO-TREX mRNA export machinery. Altogether, these studies point to a role for TINTIN, not only in co-transcriptional nucleosome recycling, but also in mRNA processing, splicing, termination and quality control. Further studies are certainly needed to confirm and dissect the function of TINTIN in relation to the processing of the mRNA molecules.

Disclosure of Potential Conflicts of Interest

J.C. holds a Canada Research Chair.

Acknowledgments

We thank Rhea Utley for comments on the text and Xue Cheng for suggesting the name TINTIN.

Funding

Work in our lab is supported by the Canadian Institutes of Health Research (Grant MOP-14308).