Recessive mutations in the ubiquitously expressed POLR3A and POLR3B genes are the most common cause of POLR3-related hypomyelinating leukodystrophy (POLR3-HLD), a rare childhood-onset disorder characterized by deficient cerebral myelin formation and cerebellar atrophy. POLR3A and POLR3B encode the two catalytic subunits of RNA Polymerase III (Pol III), which synthesizes numerous small non-coding RNAs. We recently reported that mice homozygous for the Polr3a mutation c.2015G > A (p.Gly672Glu) have no neurological abnormalities and thus do not recapitulate the human POLR3-HLD phenotype. To determine if other POLR3-HLD mutations can cause a leukodystrophy phenotype in mouse, we characterized mice carrying the Polr3b mutation c.308G > A (p.Arg103His). Surprisingly, homozygosity for this mutation was embryonically lethal with only wild-type and heterozygous animals detected at embryonic day 9.5. Using proteomics in a human cell line, we found that the POLR3B R103H mutation severely impairs assembly of the Pol III complex. We next generated Polr3aG672E/G672E/Polr3b+/R103Hdouble mutant mice but observed that this additional mutation was insufficient to elicit a neurological or transcriptional phenotype. Taken together with our previous study on Polr3a G672E mice, our results indicate that missense mutations in Polr3a and Polr3b can variably impair mouse development and Pol III function. Developing a proper model of POLR3-HLD is crucial to gain insights into the pathophysiological mechanisms involved in this devastating neurodegenerative disease.

Recessive mutations in the ubiquitously expressed POLR3A gene cause one of the most frequent forms of childhood-onset hypomyelinating leukodystrophy (HLD): POLR3-HLD. POLR3A encodes the largest subunit of RNA Polymerase III (Pol III), which is responsible for the transcription of transfer RNAs (tRNAs) and a large array of other small non-coding RNAs. In order to study the central nervous system pathophysiology of the disease, we introduced the French Canadian founder Polr3a mutation c.2015G > A (p.G672E) in mice, generating homozygous knock-in (KI/KI) as well as compound heterozygous mice for one Polr3a KI and one null allele (KI/KO). Both KI/KI and KI/KO mice are viable and are able to reproduce. To establish if they manifest a motor phenotype, WT, KI/KI and KI/KO mice were submitted to a battery of behavioral tests over one year. The KI/KI and KI/KO mice have overall normal balance, muscle strength and general locomotion. Cerebral and cerebellar Luxol Fast Blue staining and measurement of levels of myelin proteins showed no significant differences between the three groups, suggesting that myelination is not overtly impaired in Polr3a KI/KI and KI/KO mice. Finally, expression levels of several Pol III transcripts in the brain showed no statistically significant differences. We conclude that the first transgenic mice with a leukodystrophy-causing Polr3a mutation do not recapitulate the childhood-onset HLD observed in the majority of human patients with POLR3A mutations, and provide essential information to guide selection of Polr3a mutations for developing future mouse models of the disease.

The histone chaperone FACT occupies transcribed regions where it plays prominent roles in maintaining chromatin integrity and preserving epigenetic information. How it is targeted to transcribed regions, however, remains unclear. Proposed models for how FACT finds its way to transcriptionally active chromatin include docking on the RNA polymerase II (RNAPII) C-terminal domain (CTD), recruitment by elongation factors, recognition of modified histone tails and binding partially disassembled nucleosomes. Here, we systematically tested these and other scenarios in Saccharomyces cerevisiae and found that FACT binds transcribed chromatin, not RNAPII. Through a combination of experimental and mathematical modeling evidence, we propose that FACT recognizes the +1 nucleosome, as it is partially unwrapped by the engaging RNAPII, and spreads to downstream nucleosomes aided by the chromatin remodeler Chd1. Our work clarifies how FACT interacts with genes, suggests a processive mechanism for FACT function, and provides a framework to further dissect the molecular mechanisms of transcription-coupled histone chaperoning. Highlights: High-resolution mapping of FACT localization in yeast FACT binds partially unwrapped nucleosomes in transcribed genes, not RNAPII FACT distribution along genes requires Chd1 Processive mechanism for FACT function

TFIIH is an essential transcription initiation factor for RNA polymerase II (RNApII). This multi-subunit complex comprises two modules that are physically linked in Saccharomyces cerevisiae by the subunit Tfb3 (MAT1 in metazoans). The Core Module, with two DNA-dependent ATPases and several additional subunits, promotes DNA unwinding. The Kinase Module phosphorylates the C-terminal domain (CTD) of RNApII subunit Rpb1, initiating a cycle of CTD modifications that coordinate the exchange of initiation and elongation factors. Why these two disparate activities are bundled into one factor is not obvious, but the connection may provide temporal coordination during early initiation. When Tfb3 is split into two parts to uncouple the TFIIH modules, the resulting cells are viable but grow very slowly. Chromatin immunoprecipitation of the split TFIIH shows that the Core Module, but not the Kinase, is properly recruited to promoters. Instead of the normal promoter-proximal peak, high CTD Serine 5 phosphorylation is seen throughout transcribed regions. Therefore, coupling the TFIIH modules is necessary to localize and limit CTD kinase activity to early stages of transcription. These results are consistent with the idea that the two TFIIH modules began as independent functional entities that later became connected by Tfb3 during early eukaryotic evolution.

The human proteome comprises the products of more than 20,000 genes, most being arranged in complexes and networks that underlie essential cell functions. The Particle for Arrangement of Quaternary structure (PAQosome), formerly known as the R2TP/Prefoldin‐like complex, is to our knowledge the only molecular chaperoning machine designed by the evolution to assemble large protein complexes and regulatory networks in humans. The human PAQosome is a 12‐subunit complex made of two multisubunit modules, namely R2TP (RUVBL1, RUVBL2, RPAP3 and PIH1D1) and Prefoldin‐like (URI1, UXT, PDRG1, PFDN2, PFDN6 and the newly discovered ASDURF), as well as two other accessory proteins (POLR2E and WDR92). Several PAQosome clients/substrates have previously been identified, including all three nuclear RNA polymerases, various small nucleolar ribonucleoproteins (snoRNPs) and small nuclear RNPs (snRNPs), all six members of the phosphatidylinositol 3‐kinase‐related kinase (PIKK) family and others. Client specificity appears to be regulated by a number of different mechanisms, including the use of alternative core PAQosome subunits that recognize different clients directly, and the action of specificity adaptors that connect the PAQosome core to specific clients. Here, we present data identifying additional PAQosome clients, some being recruited through novel mechanisms that will be discussed. For example, and similar to what has been reported for axonemal dynein complexes involved in cilium motility, we now identify cytoplasmic dynein complexes that are responsible for cargo transport along microtubules as new clients of the PAQosome. Our recent data also indicate that specific phosphorylation of PAQosome subunits is at play for recognition of an important newly‐identified client, the pre‐ribosome, which is recruited to the PAQosome along with multiple ribosome biogenesis factors. Detailed mechanisms of assembly of some client complexes as well as the effect of mutations causing genetic diseases on complex/network integrity and PAQosome function will be presented. Characterization of the PAQosome is important not only to elucidate a newly‐recognized basal mechanism of gene regulation in eukaryotes but also to explore new research avenues that can lead to development of novel therapeutic and diagnostic tools.

Activation-induced deaminase (AID) mutates immunoglobulin genes to initiate antibody diversification by class switch recombination and somatic hypermutation but can also target non-immunoglobulin loci with oncogenic consequences. The mechanisms determining gene susceptibility to AID remain unclear. Here, we show that the H3K79 histone methyltransferase DOT1L is proximal to nuclear AID and promotes both class switch recombination and off-target AID activity, including Igh-cMyc translocations in mouse B cells. AID-mutated genes display high DOT1L activity. In the absence of DOT1L, nascent transcription largely increases despite reduced RNA Polymerase II (RNAPII) occupancy. Integrative genomic analyses reveal that DOT1L locally restricts transcription elongation velocity proportionally to H3K79me2/3 levels and extends RNAPII pausing. These transcriptional conditions enhance AID occupancy and thereby its activity. Our findings provide a harmonizing explanation for the bidirectional gene expression changes observed in DOT1L-deficient cells, and link attenuated transcriptional elongation velocity and prolonged RNAPII pausing to productive AID engagement at target loci.