Compensatory embryonic response to allele-specific inactivation of the murine X-linked gene Hcfc1

Abstract

Early in female mammalian embryonic development, cells randomly inactivate one of the two X chromosomes to achieve overall equal inactivation of parental X-linked alleles. Hcfc1 is a highly conserved X-linked mouse gene that encodes HCF-1 - a transcriptional co-regulator implicated in cell proliferation in tissue culture cells. By generating a Cre-recombinase inducible Hcfc1 knock-out (Hcfc1(lox)) allele in mice, we have probed the role of HCF-1 in actively proliferating embryonic cells and in cell-cycle re-entry of resting differentiated adult cells using a liver regeneration model. HCF-1 function is required for both extraembryonic and embryonic development. In heterozygous Hcfc1(lox/+) female embryos, however, embryonic epiblast-specific Cre-induced Hcfc1 deletion (creating an Hcfc1(epiKO) allele) around E5.5 is well tolerated; it leads to a mixture of HCF-1-positive and -negative epiblast cells owing to random X-chromosome inactivation of the wild-type or Hcfc1(epiKO) mutant allele. At E6.5 and E7.5, both HCF-1-positive and -negative epiblast cells proliferate, but gradually by E8.5, HCF-1-negative cells disappear owing to cell-cycle exit and apoptosis. Although generating a temporary developmental retardation, the loss of HCF-1-negative cells is tolerated, leading to viable heterozygous offspring with 100% skewed inactivation of the X-linked Hcfc1(epiKO) allele. In resting adult liver cells, the requirement for HCF-1 in cell proliferation was more evident as hepatocytes lacking HCF-1 fail to re-enter the cell cycle and thus to proliferate during liver regeneration. The survival of the heterozygous Hcfc1(epiKO/+) female embryos, even with half the cells genetically compromised, illustrates the developmental plasticity of the post-implantation mouse embryo - in this instance, permitting survival of females heterozygous for an X-linked embryonic lethal allele.

Cortical and Commissural Defects Upon HCF‐1 Loss in Nkx2.1‐Derived Embryonic Neurons and Glia

“…Fig. 1A1) (Minocha et al , ), but with a progressive reduction in relative HCF‐1 protein levels with age (Supp. Fig.…”

“…The human HCFC1 and mouse Hcfc1 genes are highly expressed in actively dividing tissue culture cells, and in embryonic and placental tissues and in adult tissues (Wilson et al , ; Frattini et al , ; Kristie, ; Huang et al , ; Minocha et al , ). Previously, using a well‐characterized antibody generating little to no non‐specific reactivity (H12), we have shown that HCF‐1 is ubiquitous and predominantly nuclear in E6.5‐to‐E12.5 embryos, postnatal day 0 (P0) brains, and 10‐week‐old young adult brains (Minocha et al , ). Here, we further investigated in detail the cellular and subcellular localization of HCF‐1 in the early mouse brain by immunostaining wild‐type C57BL/6 postnatal day 0 (P0) brains.…”

“…We induced conditional deletion of the Hcfc1 gene in these cell types by crossing males heterozygous for an Nkx2.1‐Cre transgene (referred to as Nkx2.1‐Cre + ) (Xu et al , ) with Hcfc1 lox/lox females (Minocha et al , ). The X‐linked Hcfc1 lox allele contains two loxP sites, one in intron 1 and another in intron 3 that undergo recombination in the presence of Cre recombinase, deleting exons 2 and 3 to generate the conditional knockout (cKO) allele encoding a highly truncated 66 amino acid long N‐terminal HCF‐1 protein (Minocha et al , ). Thus, hemizygous Hcfc1 lox/Y males carrying the Nkx2.1‐Cre + allele were expected to generate a complete embryonic Nkx2.1 ‐specific knock out.…”

“…PCR genotyping of these lines was performed as described previously (Niquille et al , ). We used Hcfc1 lox/lox (Minocha et al , ), Nkx2.1 ‐ Cre (Xu et al , ), and GLAST‐Cre:ERT2 (The Jackson Laboratory, Bar Harbor, Maine, USA, Tg(Slc1a3‐cre/ERT)1Nat/J)) (Minocha et al , ) transgenic mice described previously. The reporter Rosa26R–GFP mouse line was used to reliably express GFP under the control of the Rosa26 promoter upon Cre‐mediated recombination.…”

“…Hcfc1 encodes HCF‐1, a conserved transcriptional co‐regulator that binds to the transcriptional start sites of many genes (Dejosez et al , ; Michaud et al , ) and associates with both sequence‐specific DNA‐binding proteins (e.g., Myc, E2F1 and E2F4, Thap11/Ronin, ZNF143) and chromatin‐modifying enzymes (e.g., the MLL and Set1 histone H3 Lysine 4 methyltransferases and Sin3 histone deacetylase) (reviewed in (Zargar and Tyagi, ); see also (Thomas et al , )). Genetic studies in mammalian cell culture, early mouse embryos, and liver have shown that HCF‐1 is important for multiple aspects of cell proliferation (Goto et al , ; Reilly and Herr, ; Julien and Herr, ; Minocha et al , ), and very early epiblast‐specific embryonic loss of HCF‐1 is lethal before gastrulation (Minocha et al , ; ). Nevertheless, in humans, there are mutations in the HCFC1 gene that are associated with X‐linked intellectual disability (ID) and cobalamin metabolism; these disorders point toward an important role of HCF‐1 in brain development (Huang et al , ; Yu et al , ; Gerard et al , ; Jolly et al , ; Koufaris et al , ).…”