GEMC1 and MCIDAS interactions with SWI/SNF complexes regulate the multiciliated cell-specific transcriptional program.

Multiciliated cells (MCCs) project dozens to hundreds of motile cilia from their apical surface to promote the movement of fluids or gametes in the mammalian brain, airway or reproductive organs. Differentiation of MCCs requires the sequential action of the Geminin family transcriptional activators, GEMC1 and MCIDAS, that both interact with E2F4/5-DP1. How these factors activate transcription and the extent to which they play redundant functions remains poorly understood. Here, we demonstrate that the transcriptional targets and proximal proteomes of GEMC1 and MCIDAS are highly similar. However, we identified distinct interactions with SWI/SNF subcomplexes; GEMC1 interacts primarily with the ARID1A containing BAF complex while MCIDAS interacts primarily with BRD9 containing ncBAF complexes. Treatment with a BRD9 inhibitor impaired MCIDAS-mediated activation of several target genes and compromised the MCC differentiation program in multiple cell based models. Our data suggest that the differential engagement of distinct SWI/SNF subcomplexes by GEMC1 and MCIDAS is required for MCC-specific transcriptional regulation and mediated by their distinct C-terminal domains.

GEMC1 is a critical regulator of multiciliated cell differentiation.

The generation of multiciliated cells (MCCs) is required for the proper function of many tissues, including the respiratory tract, brain, and germline. Defects in MCC development have been demonstrated to cause a subclass of mucociliary clearance disorders termed reduced generation of multiple motile cilia (RGMC). To date, only two genes, Multicilin (MCIDAS) and cyclin O (CCNO) have been identified in this disorder in humans. Here, we describe mice lacking GEMC1 (GMNC), a protein with a similar domain organization as Multicilin that has been implicated in DNA replication control. We have found that GEMC1-deficient mice are growth impaired, develop hydrocephaly with a high penetrance, and are infertile, due to defects in the formation of MCCs in the brain, respiratory tract, and germline. Our data demonstrate that GEMC1 is a critical regulator of MCC differentiation and a candidate gene for human RGMC or related disorders.

Sensing of replication stress and Mec1 activation act through two independent pathways involving the 9-1-1 complex and DNA polymerase ε.

Following DNA damage or replication stress, budding yeast cells activate the Rad53 checkpoint kinase, promoting genome stability in these challenging conditions. The DNA damage and replication checkpoint pathways are partially overlapping, sharing several factors, but are also differentiated at various levels. The upstream kinase Mec1 is required to activate both signaling cascades together with the 9-1-1 PCNA-like complex and the Dpb11 (hTopBP1) protein. After DNA damage, Dpb11 is also needed to recruit the adaptor protein Rad9 (h53BP1). Here we analyzed the mechanisms leading to Mec1 activation in vivo after DNA damage and replication stress. We found that a ddc1Δdpb11-1 double mutant strain displays a synthetic defect in Rad53 and H2A phosphorylation and is extremely sensitive to hydroxyurea (HU), indicating that Dpb11 and the 9-1-1 complex independently promote Mec1 activation. A similar phenotype is observed when both the 9-1-1 complex and the Dpb4 non-essential subunit of DNA polymerase ε (Polε) are contemporarily absent, indicating that checkpoint activation in response to replication stress is achieved through two independent pathways, requiring the 9-1-1 complex and Polε.

GEMC1 is a novel TopBP1-interacting protein involved in chromosomal DNA replication.

Chromosomal DNA must be precisely replicated in each cell cycle in order to ensure maintenance of genome stability. Most of the factors controlling this process have been identified in lower eukaryotes. Several factors involved in DNA replication are also important for the cellular response to stress conditions. However, the regulation of DNA replication in multi-cellular organisms is still poorly understood. Using the Xenopus laevis egg cell-free system, we have recently identified a novel vertebrate protein named GEMC1 required for DNA replication. xGEMC1 is a Cyclin dependent kinase (CDK) target required forCdc45 loading onto chromatin and it interacts with the checkpoint and replication factor TopBP1, which promotes its binding to chromatin during prereplication complex formation. Here we discuss our recent findings and propose possible roles for GEMC1. Interestingly, recent studies have identified other proteins with analogous functions, showing a higher level of complexity in metazoan replication control compared to lower eukaryotes.

Phosphorylation of the budding yeast 9-1-1 complex is required for Dpb11 function in the full activation of the UV-induced DNA damage checkpoint.

Following genotoxic insults, eukaryotic cells trigger a signal transduction cascade known as the DNA damage checkpoint response, which involves the loading onto DNA of an apical kinase and several downstream factors. Chromatin modifications play an important role in recruiting checkpoint proteins. In budding yeast, methylated H3-K79 is bound by the checkpoint factor Rad9. Loss of Dot1 prevents H3-K79 methylation, leading to a checkpoint defect in the G(1) phase of the cell cycle and to a reduction of checkpoint activation in mitosis, suggesting that another pathway contributes to Rad9 recruitment in M phase. We found that the replication factor Dpb11 is the keystone of this second pathway. dot1Delta dpb11-1 mutant cells are sensitive to UV or Zeocin treatment and cannot activate Rad53 if irradiated in M phase. Our data suggest that Dpb11 is held in proximity to damaged DNA through an interaction with the phosphorylated 9-1-1 complex, leading to Mec1-dependent phosphorylation of Rad9. Dpb11 is also phosphorylated after DNA damage, and this modification is lost in a nonphosphorylatable ddc1-T602A mutant. Finally, we show that, in vivo, Dpb11 cooperates with Dot1 in promoting Rad9 phosphorylation but also contributes to the full activation of Mec1 kinase.