Congenital heart defects often result from improper differentiation of cardiac progenitor cells. Although transcription factors involved in cardiac progenitor cell differentiation have been described, the associated chromatin modifiers in this process remain largely unknown. Here we show that mouse embryos lacking the chromatin-modifying enzyme histone deacetylase 3 (Hdac3) in cardiac progenitor cells exhibit precocious cardiomyocyte differentiation, severe cardiac developmental defects, upregulation of Tbx5 target genes and embryonic lethality. Hdac3 physically interacts with Tbx5 and modulates its acetylation to repress Tbx5-dependent activation of cardiomyocyte lineage-specific genes. These findings reveal that Hdac3 plays a critical role in cardiac progenitor cells to regulate early cardiogenesis.

There is variability as to how archaea catalyze the final step of de novo purine biosynthesis to form inosine 5’-monophosphate (IMP) from 5-formamidoimidazole-4-carboxamide ribonucleotide (FAICAR). Although non-archaea almost uniformly use the bifunctional PurH protein, which has an N-terminal IMP cyclohydrolase (PurH2) fused to a C-terminal folate-dependent aminoimidazole-4-carboxamide ribonucleotide (AICAR) formyltransferase (PurH1) domain, a survey of the genomes of archaea reveals use of PurH2 (with or without fusion to PurH1), the “euryarchaeal signature protein” PurO, or an unidentified crenarchaeal IMP cyclohydrolase. In this report, we present the cloning and functional characterization of two representatives of the known IMP cyclohydrolase families. The locus TK0430 in Thermococcus kodakarensis encodes a PurO-type IMP cyclohydrolase with demonstrated activity despite its position in a cluster of apparently redundant biosynthetic genes, the first functional characterization of a PurO from a non-methanogen. Kinetic characterization reveals a Km for FAICAR of 1.56 ± 0.39 μM and a kcat of 0.48 ± 0.04 s-1. The locus AF1811 from Archaeoglobus fulgidus encodes a PurH2-type IMP cyclohydrolase. This Archaeoglobus fulgidus PurH2 has a Km of 7.8 ± 1.8 μM and kcat of 1.32 ± 0.14 s-1, representing the first characterization of an archaeal PurH2 and the first characterization of PurH2 that naturally occurs unfused to an AICAR formyltransferase domain. Each of these two characterized IMP cyclohydrolases converts FAICAR to IMP in vitro, and each cloned gene allows the growth on purine-deficient media of an E. coli purine auxotroph lacking the purH2 gene.

There is some diversity as to how archaea effect the final step of de novo purine biosynthesis to form inosine 5′‐monophosphate (IMP) from 5‐formamidoimidazole‐4‐carboxamide ribonucleotide (FAICAR). A survey of the genomes of archaea reveals a mixture of PurH2 (with or without fusion to PurH1), the Euryarchaeal signature protein PurO, and an unidentified Crenarchaeal IMP cyclohydrolase. In this report, we present the cloning and functional characterization of three representatives of the known IMP cyclohydrolases. The locus TK0430 in Thermococcus kodakarensis encodes a PurO‐type IMP cyclohydrolase with demonstrated activity despite its position in a cluster of apparently redundant biosynthetic genes. The locus AF1811 from Archaeoglobus fulgidus encodes a PurH2‐type IMP cyclohydrolase that occurs in active form without fusion to a PurH1 protein. The locus HVO_0011 in Haloferax volcanii encodes a PurO‐type IMP cyclohydrolase active only at high salt concentrations. All three enzymes convert FAICAR to IMP in vitro, and two restore the growth on purine deficient media of an E. coli purine auxotroph lacking purH2.

This work is a survey of known and suspected purine biosynthesis genes in sequenced archaea. We searched existing sequence databases for genes for the 11 steps (17 distinct genes) of de novo purine biosynthesis in 65 sequenced archaea, and carefully inspected each hit for the presence of active site residues and other residues known or suspected to be required for function. We found a number of problems with existing annotations, such as missing active site residues, fusions, and incorrect initiation sites. There is also evidence that the pathway has been shaped by horizontal gene transfer, duplication, and gene loss. Two groups of archaea lack a gene normally required for purine biosynthesis, suggesting that there are at least two more variants in this pathway to discover. We propose the existence of a gene encoding a GAR transformylase enzyme that is structurally unrelated to either known GAR transformylase, expected to be present in seven of the 65 archaea studied. A second proposed gene encodes a novel IMP cyclohydrolase function and is expected to be present in the Crenarchaea and Pyrococci.Functional characterization of some purine biosynthesis enzymes will also be reported.

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