Taspase1 orchestrates fetal liver hematopoietic stem cell and vertebrae fates by cleaving TFIIA

Taspase1, a highly conserved threonine protease encoded by TASP1, cleaves nuclear histone-modifying factors and basal transcription regulators to orchestrate diverse transcription programs. Hereditary loss-of-function mutation of TASP1 has recently been reported in humans as resulting in an anomaly complex syndrome, which manifests with hematological, facial, and skeletal abnormalities. Here, we demonstrate that Taspase1-mediated cleavage of TFIIAα-β, rather than of MLL1 or MLL2, in mouse embryos was required for proper fetal liver hematopoiesis and correct segmental identities of the axial skeleton. Homozygous genetic deletion of Taspase1 disrupted embryonic hematopoietic stem cell self-renewal and quiescence states and axial skeleton fates. Strikingly, mice carrying knockin noncleavable mutations of TFIIAα-β, a well-characterized basal transcription factor, displayed more pronounced fetal liver and axial skeleton defects than those with noncleavable MLL1 and MLL2, 2 trithorax group histone H3 trimethyl transferases. Our study offers molecular insights into a syndrome in humans that results from loss of TASP1 and describes an unexpected role of TFIIAα-β cleavage in embryonic cell fate decisions.

Hereditary loss-of-function mutation of TASP1 has recently been reported in humans as resulting in an anomaly complex syndrome, which manifests with hematological, facial, and skeletal abnormalities. Here, we demonstrate that Taspase1-mediated cleavage of TFIIAα-β, rather than of MLL1 or MLL2, in mouse embryos was required for proper fetal liver hematopoiesis and correct segmental identities of the axial skeleton. Homozygous genetic deletion of Taspase1 disrupted embryonic hematopoietic stem cell self-renewal and quiescence states and axial skeleton fates. Strikingly, mice carrying knockin noncleavable mutations of TFIIAα-β, a well-characterized basal transcription factor, displayed more pronounced fetal liver and axial skeleton defects than those with noncleavable MLL1 and MLL2, 2 trithorax group histone H3 trimethyl transferases. Our study offers molecular insights into a syndrome in humans that results from loss of TASP1 and describes an unexpected role of TFIIAα-β cleavage in embryonic cell fate decisions.
Taspase1-mediated cleavage of each substrate may account for various phenotypes of Taspase1 deficiency, or, alternatively, protease-independent biological activities of Taspase1 may exist. Here, we performed mouse genetic studies to interrogate the biological significance of cleavage of MLL1, MLL2, and TFIIA. Taspase1-mediated cleavage of TFIIA has the most prominent effects on fetal hematopoiesis as well as on the specification of axial skeleton, offering molecular insights concerning a syndrome in humans that results from loss of TASP1.

Results
To investigate the physiological function of Taspase1, we generated Tasp1-deficient mice (Tasp1 -/mice). Our initial report demonstrated that these animals displayed marked homeotic transformation of the axial skeleton and a decrease in overall body size (11). Furthermore, Tasp1 -/mice die shortly after birth, partly due to feeding defects (11). Here, we further examined the role of Taspase1 in embryogenesis and initiated studies by comparing these Tasp1 -/and WT littermate embryos. Tasp1 -/-E14.5 embryos show a severe developmental retardation, reflected by an approximately 40% reduction in overall weight in comparison to that of WT embryos ( Figure 1A). Furthermore, Tasp1 -/embryos have markedly smaller fetal livers ( Figure  1B). The decrease in fetal liver cell number was significant even after normalizing for reduced body weight (P < 0.001) ( Figure 1A).
We next analyzed the fetal liver for mature blood cell lineages and the hematopoietic stem and progenitor cell compartments using multicolor flow cytometry. The analysis of E14.5 fetal livers showed that Tasp1 -/and WT fetal livers had little difference in the populations of B220 + B cells, Gr-1 + granulocytes, and TER-119 + red blood cells ( Figure 1C). In contrast, the stem and progenitor cell populations, marked by Lin -Sca-1 + c-Kit + staining (so-called LSK cells), were reduced in Tasp1 -/fetal livers (Figure 1, D and E). LSK cells can be further specified as multipotent progenitor cells (MPPs; Lin -Sca-1 + c-Kit + CD150 -) or hematopoietic stem cells (HSCs; Lin -Sca-1 + c-Kit + CD150 + ) (24,25). In Tasp1 -/fetal livers, the abundance of HSCs was found to be reduced to approximately half that of the WT fetal liver ( Figure 1E), resulting in an approximately 78% decrease in the absolute number of HSCs ( Figure 1F). No difference in the frequency of Lin -Sca-1c-Kit + myeloid progenitors was observed, whereas absolute numbers of these cells were decreased. Specifically, in Tasp1 -/fetal livers, the relative abundance of common myeloid progenitors (CMPs), granulocyte-monocyte progenitors (GMPs), and megakaryocyte-erythrocyte progenitors (MEPs) was normal ( Figure 1, D and E). Together, our data indicate that, in the mouse fetal liver, Taspase1 may be required for the development and maintenance of the HSC compartment.
Accordingly, we investigated how Taspase1 loss leads to impaired fetal liver HSC renewal. Loss of Taspase1 activity could lead to increased cell death and/or aberrant cell cycle control. Cell death analysis of LSK cells and HSCs showed no increase of apoptosis in Tasp1 -/fetal livers ( Figure 2A). On the other hand, cell cycle analysis of MPPs and HSCs yielded a marked difference between WT and Tasp1 -/cells. Most notably staining of DNA and RNA with 7-AAD and pyronin Y, respectively, showed that more Tasp1 -/fetal liver HSCs exited stem cell quiescence, with decreased number of cells in the G 0 phase, whereas the majority of WT HSCs were in the G 0 phase, with fewer cells at G 1 and S/G 2 /M ( Figure 2, B and C). In contrast, Tasp1 -/fetal liver MPPs displayed similar cell cycle profile as WT MPPs. Strikingly, the cell cycle profile observed of Tasp1 -/fetal liver HSCs resembles that of MPPs ( Figure 2B).
To elucidate by which substrate(s) cleavage of Taspase1 regulates fetal liver hematopoiesis, we first focused on the MLL family proteins MLL1 and MLL2. MLL1 is the best-characterized Taspase1 substrate, controls Hox and Cylclin gene expression, and plays a critical role in both fetal and adult hematopoiesis (26)(27)(28). Homozygous knockin of noncleavable (nc) Mll1 mutant alleles (Mll1 nc/nc ) at the endogenous Mll1 locus did not reduce the overall fetal liver cellularity ( Figure 3A) but incurred a minor decrease in frequency and number of HSCs ( Figure 3B). As published data suggest a partial redundancy between Mll1 and Mll2 (11), we analyzed E14.5 fetal livers of Mll1 nc/nc ;2 nc/nc embryos bearing homozygous knockin of nc Mll1 and nc Mll2 mutant alleles at their native genomic loci and did not detect reduced fetal liver cellularity or body weight ( Figure 3C). Similarly, the reduced abundance or absolute number of HSCs in Mll1 nc/nc ;2 nc/nc fetal livers was not statistically significant ( Figure 3D).
TFIIA family proteins TFIIAα-β and ALF are the only bona fide mammalian Taspase1 substrates known thus far besides MLL1 and MLL2. As expression of ALF is restricted to the mammalian testis (29) we focused on the noncleavage of TFIIAα-β. We have created a Gtf2a1 nc/nc mouse model in which the endogenous D/G cleavage residues of TFIIAα-β are replaced with noncleavable A/A and have reported that Gtf2a1 nc/nc males are infertile (22). Remarkably, Gtf2a1 nc/nc embryos were smaller than their WT littermates, and their livers were disproportionally smaller ( Figure 4, A and B). Furthermore, the frequencies of HSCs in Gtf2a1 nc/nc fetal livers were also reduced ( Figure 4D). In fact, the absolute HSC number of Gtf2a1 nc/nc fetal livers was approximately 30% of that of their WT littermates ( Figure 4E). Similar to Tasp1 -/embryos, there were no significant changes in the frequency of myeloid progenitors, CMPs, GMPs, or MEPs in Gtf2a1 nc/nc fetal livers (Figure 4, C and D). Cell cycle analysis of Gtf2a1 nc/nc fetal liver stem and progenitor cells demonstrated decreased a number of Gtf2a1 nc/nc HSCs in the G 0 phase, which is reminiscent to that of WT and Gtf2a1 nc/nc MPPs ( Figure 5). The decrease in G 0 phase and the coinciding increases in non-G 0 phases of the Gtf2a1 nc/nc fetal liver HSCs indicated a stem cell quiescence defect similar to that observed in Tasp1 -/fetal liver HSCs (Figure 2, B and C). These striking similarities between Gtf2a1 nc/nc and Tasp1 -/fetal livers highlight the importance of the Taspase1-TFIIA axis in the maintenance of fetal liver HSCs.
To further investigate this potentially novel regulation, we employed stem cell transplant assays to evaluate the capacity of Tasp1 -/and Gtf2a1 nc/nc fetal liver HSCs in long-term hematopoietic reconstitution. Competitive repopulation assays were performed by transplanting 150 CD45.2 + HSCs from WT, Tasp1 -/-, or Gtf2a1 nc/nc E14.5 fetal livers along with CD45.1 + competitor cells into lethally irradiated CD45.1 + mice (Figure 6A). Twelve weeks after transplantation, peripheral blood was analyzed for the contribution of donor-derived HSCs to mature blood lineages, including B220 + for B cells, CD3 + for T cells, and Gr-1 + Mac-1 + for myeloid cells (Figure 6, B and C). More than 5% of CD45.2 + donor-derived cells in all 3 lineages were detected in 13 of 19 mice transplanted with WT HSCs, whereas multilineage reconstitution was detected in neither the 9 mice transplanted with Tasp1 -/-HSCs nor the 8 mice transplanted with Gtf2a1 nc/nc HSCs (Table  1). Thus, the Taspase1-TFIIA axis appears to be required for long-term reconstitution of hematopoietic cells from fetal livers, suggesting its role in long-term HSC self-renewal.
The phenotypic similarities between Gtf2a1 nc/nc and Tasp1 -/fetal liver HSCs indicate that TFI-IA is the principal Taspase1 substrate conferring Taspase1-orchestrated fetal liver hematopoiesis. As noncleaved TFIIAα-β nc is more stable than cleaved TFIIA (5,22) and no apparent abnormalities were detected in Gtf2a1 nc/+ fetal livers (data not shown), TFIIAα-β nc is unlikely to function as a dominant-negative mutant in fetal liver hematopoiesis. Instead, data favors cleaved TFIIA positively regulating fetal liver hematopoiesis.
Hox genes of the vertebrates and homeotic genes of the invertebrates play critical roles in implementing body plan, and their deregulation results in the loss of segmental identities, i.e., homeotic transformation. As our previous studies demonstrated that Tasp1 -/mice exhibit homeotic transformation (11), we examined the axial skeleton of Gtf2a1 nc/nc newborn pups (n = 17) and detected overt homeotic  Table 2). Unexpectedly, homeotic defects of Gtf2a1 nc/nc newborns were more profound than those of Mll1 nc/nc ;2 nc/nc newborns and highly reminiscent of those of Tasp1 -/newborns ( Figure 7 and Table 2). Homeotic transformation of the axial skeletons is the defining feature of Hox gene deregulation (30,31), and these extensive homeotic defects in Gtf2a1 nc/nc mice indicate a potentially novel regulation of Hox genes by TFIIA, a basal transcription factor, through a site-specific proteolytic process.

Discussion
Unlike most reversible posttranslational protein modifications, such as phosphorylation, acetylation, and methylation, proteolysis through either degradation or site-specific cleavage renders structural changes permanent and, thereby, potentially results in long-lasting functional consequences. In metazoans, site-specific proteolysis regulates critical aspects of biology, such as the activation of blood coagulation factors for hemostasis, the activation of caspases for cell death execution, the cleavage of Notch intracellular domain for cell fate determination, the release of SREBP for cholesterol homeostasis (32), the maturation of HCF and MLL1 for cell cycle progression (11,12), and the assembly of mature TFIIAα/β/γ for male germ cell maturation (5,22). Indeed, the identification and functional characterization of proteases and their cognate substrates have been instrumental in unraveling the underlying mechanisms concerning diverse biological processes.
Taspase1 is a highly conserved protease that orchestrates a plethora of genetic programs through cleaving nuclear transcription regulators, MLL1, MLL2, TFIIA, ALF, and dHCF (6,33). Given that TFIIA is a basal transcription factor and Hox genes are highly specialized transcription factors, the connections between Taspase1-mediated proteolytic processing of TFIIA and transcriptional regulation of Hox genes were completely unexpected, adding an additional layer of complexity to the intricate control of Hox gene expression through upstream transcription factors, epigenetic regulators, and long intergenic noncoding RNA (34,35) for constructing segmental body plan and specifying cell lineages including stem cells.  The paradigm of Taspase1 function provides an opportunity to investigate how proteases interconnect diverse genetic programs via site-specific proteolysis. In the absence of Taspase1-mediated cleavage, MLL1, MLL2, and TFIIAα-β retain partial activity and animals bearing noncleaved MLL1, MLL2, and/or TFIIAα-β manifest hypomorphism in several biological settings (11,22,36,37). Consequently, the alleles encoding nc MLL1, MLL2, and TFIIAα-β differ from null alleles and thus provide invaluable insight in cases where genetic ablation results in early embryonic lethality. Likewise, Taspase1 deficiency appears to offer a unique opportunity to uncover signaling pathways  controlled by developmentally essential genes. Taspase1 regulates the cell cycle, axial skeletal formation, fetal HSC homeostasis, male germ cell development (11,22,36,37), and craniofacial development (23). As TFIIA complexes with not only TBP but also TRF2 (a male germ cell-enriched TBP variant) and TRF3 (a vertebrate-specific TBP variant), the Taspase1-TFIIA axis is positioned to orchestrate the assembly of diverse tissue-specific or context-dependent transcription machinery that are crucial for organismal development.
Sorting of hematopoietic stem and progenitor cells. Single-cell suspensions were prepared from fetal livers of E14.5 embryos or adult bone marrow and stained as described above. Fetal liver cells from multiple littermate embryos of the same genotype were pooled to ensure sufficient number of cells for staining. Stained cells were subsequently double sorted using a FACSAria II (BD Biosciences) to a final purity of more than 95%.
Cell cycle analysis. Suspensions of sorted hematopoietic stem and progenitor cells were fixed with ethanol overnight at 4°C, washed, and then resuspended in the house-made Nucleic Acid Staining Solution (38). Cells were stained with 7-AAD at room temperature for 20 minutes, followed by pyronin Y staining on ice for 15 minutes. Stained cells were analyzed by a LSRFortessa flow cytometer.
Statistics. Statistical significance was evaluated using the Mann-Whitney U test for continuous variables, unless otherwise specified. χ 2 testing was performed on cell cycle analyses, and P values of independent experiments were combined by the Fisher's method. P values of less than 0.05 were considered significant.
Study approval. All animal work was performed in accordance to a protocol approved by the Institutional Animal Care and Use Committee of Memorial Sloan Kettering Cancer Center.