Kleefstra syndrome, caused by haploinsufficiency of euchromatin histone methyltransferase 1 (EHMT1), is characterized by intellectual disability (ID), autism spectrum disorder (ASD), characteristic facial dysmorphisms, and other variable clinical features. In addition to EHMT1 mutations, de novo variants were reported in four additional genes (MBD5, SMARCB1, NR1I3, and KMT2C), in single individuals with clinical characteristics overlapping Kleefstra syndrome. Here, we present a novel cohort of five patients with de novo loss of function mutations affecting the histone methyltransferase KMT2C. Our clinical data delineates the KMT2C phenotypic spectrum and reinforces the phenotypic overlap with Kleefstra syndrome and other related ID disorders. To elucidate the common molecular basis of the neuropathology associated with mutations in KMT2C and EHMT1, we characterized the role of the Drosophila KMT2C ortholog, trithorax related (trr), in the nervous system. Similar to the Drosophila EHMT1 ortholog, G9a, trr is required in the mushroom body for short term memory. Trr ChIP-seq identified 3371 binding sites, mainly in the promoter of genes involved in neuronal processes. Transcriptional profiling of pan-neuronal trr knockdown and G9a null mutant fly heads identified 613 and 1123 misregulated genes, respectively. These gene sets show a significant overlap and are associated with nearly identical gene ontology enrichments. The majority of the observed biological convergence is derived from predicted indirect target genes. However, trr and G9a also have common direct targets, including the Drosophila ortholog of Arc (Arc1), a key regulator of synaptic plasticity. Our data highlight the clinical and molecular convergence between the KMT2 and EHMT protein families, which may contribute to a molecular network underlying a larger group of ID/ASD-related disorders.

Many children with the CHARGE association have facial paralysis and feeding and swallowing difficulties. Indeed, facial paralysis and pharyngeal incoordination may be important diagnostic indicators of CHARGE association [Davenport et al., 1986a; Mitchell et al., 1985]. We describe an individual with dysfunction of multiple cranial nerves (Möbius sequence) and CHARGE association, a previously unrecognized combination. Our review of 150 patients from the literature and 13 patients from this center with CHARGE association documented that dysfunction of cranial nerves is frequent in CHARGE association, and that often cranial nerve abnormalities are multiple. Such multiple cranial nerve abnormalities may be the primary underlying cause for the facial paralysis, feeding difficulties and sensorineural hearing loss seen in many individuals with CHARGE association.

Transcription of the metH gene in Salmonella typhimurium and Escherichia coli is positively regulated by the metR gene product, a DNA binding protein. The interaction between the MetR activator protein and the S. typhimurium metH control region was investigated. In vitro gel mobility shift assays and DNase I protection assays established that the MetR protein binds to and protects a 24-bp sequence in the metH promoter region from DNase I attack. This region includes the proposed metR recognition sequence 5'-TGAANNNNNCTCA-3'. Single-base-pair changes were introduced into the proposed MetR recognition sequence within the promoter region of a metH-lacZ gene fusion by oligonucleotide-directed mutagenesis. Two classes of mutations were identified. In the first class, the mutations caused reduced activation of the metH-lacZ fusions that correlated with reduced MetR binding. In the second class, activation of the metH-lacZ fusion was reduced, yet there was no appreciable reduction in MetR binding, indicating that the presence of bound MetR is not sufficient for activation of metH-lacZ gene expression. These two classes of mutations in the DNA binding site are grouped spatially, suggesting that the proposed MetR recognition sequence can be divided into two functional domains, one for binding and the other for activation.

Using an Escherichia coli lac deletion strain lysogenized with a A phage carrying a metH-lacZ gene fusion, we isolated trans-acting mutations that result in simultaneous 4-to 6-fold-elevated metH-lacZ expression, 5-to 22-fold-lowered metE-lacZ expression, and 9-to 20-fold-elevated metR-lacZ expression. The altered regulation of these genes occurs in the presence of high intracellular levels of homocysteine, a methionine pathway intermediate which normally inhibits metH and metR expression and stimulates metE expression. P1 transductions and complementation tests indicate that the mutations are in the metR gene. Our data suggest that the mutations result in an altered MetR activator protein that has lost the ability to use homocysteine as a modulator of gene expression.In Escherichia coli and Salmonella typhimurium the methylation of homocysteine to form methionine is catalyzed by either the or the MetH (vitamin B12-dependent) transmethylase (for a review, see reference 11). This reaction links the serine-glycine and methionine biosynthetic pathways (Fig. 1). The cell controls expression of the genes encoding the methionine biosynthetic enzymes by both negative and positive regulatory mechanisms. The MetJ repressor, with S-adenosylmethionine acting as a corepressor, negatively regulates all of the methionine biosynthetic genes, with the exception of metH (11). The MetR gene product, a DNA-binding protein (2, 18), is necessary for the activation of both metE and metH expression (2,20). Homocysteine modulates this activation by stimulating metE expression and inhibiting metH expression (19). The MetJ repressor system, however, can override activation by the MetR protein (20). The metR gene itself is negatively autoregulated, with homocysteine acting as the corepressor (16).Using a XmetH-lacZ fusion phage in an E. coli strain that accumulates high endogenous levels of homocysteine, we isolated mutants in which homocysteine no longer inhibits metH-lacZ expression. Using metH-lacZ, metE-lacZ, and metR-lacZ gene fusions to measure expression of the metH, metE, and metR genes, respectively, we found that the mutations affect the regulation of all three genes. Our data suggest that the mutations lie in the metR gene, altering the ability of the MetR activator protein to use homocysteine as a modulator of gene expression.MATERIALS AND METHODS Bacterial strains, bacteriophages, and plasmids. All bacterial strains used were derived from E. coli K-12 and are described in Table 1. Construction of the lacZ fusion phages XElac (10), XHlac (17), XRlac (16), and XRElac (19) has been described previously. Plasmid pGSmetR (16) carries the S. typhimurium metR gene on the single-copy vector pDF41 (6).Media and growth conditions. Luria agar (L agar), Luria broth (LB), and glucose minimal medium (GM) have been described previously (13). Lactose minimal medium (LM) was identical to GM, except that lactose replaced glucose. * Corresponding author.GM and LM were always supplemented with phenylalanine (50 ,ug/ml) and vitamin B1 (1 jig/ml)...

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