Spacing Constraints of Neighboring Zinc Finger Modules within GATA2

Genomic analyses in clinical and experimental contexts have accelerated discoveries of human genetic variants. While elucidating the consequences of conspicuously loss-of-function variants is highly tractable, decoding the impact of missense or non-coding variants is considerably more challenging. Previously, we described a germline variant in GATA2 in a patient with GATA2-deficiency syndrome, which inserts nine amino acids between the two zinc fingers (9aa-Ins), one of which mediates sequence-specific DNA binding (Cavalcante de Andrade Silva M. et al., Leukemia, 2021). Unlike other GATA2 coding region and enhancer variants identified (Bresnick E.H. et al., Blood Adv., 2020), it was unclear whether the altered zinc finger spacing would be inhibitory, stimulatory or of no consequence. The 9aa-Ins variant was defective in activating several target genes (Hdc, Ear2 and Tpsb2) in a genetic complementation assay with Gata2 -77 enhancer-mutant (-77 -/-) primary hematopoietic progenitor cells. As only several target genes were tested, we used RNA-seq to conduct a genome-wide comparison of the capacity of GATA2 and 9aa-Ins to activate and repress transcription. To elucidate mechanisms, we considered the following models: 1) 9aa-Ins fails to regulate all genes normally controlled by GATA2; 2) 9aa-Ins fails to repress all genes normally controlled by GATA2; 3) 9aa-Ins fails to activate genes normally controlled by GATA2; 4) 9aa-Ins ectopically regulates genes not controlled by GATA2. Using a genetic complementation approach with -77 -/- cells that were immortalized by the Hoxb8 transcription factor (hi-77 -/-) (Wang G.G. et al., Nat. Methods, 2006; Johnson K.D. et al., JEM, 2020), we compared GATA2 and 9aa-Ins activities when expressed at a comparable level. This analysis revealed 2,138 GATA2-regulated, 525 GATA2 and 9aa-Ins-regulated, and 414 ectopically-regulated genes (at least two-fold change, adjusted P-value <0.05). A similar number of genes were GATA2-activated (1,061) and repressed (1,077). Only 144 out of the 1,061 (14%) were 9aa-Ins-activated and 381 out of 1,077 (35%) were 9aa-Ins-repressed, illustrating the severe consequences of this mutation and a greater impact on activation versus repression. Statistical analysis with a range of P-values constraints (0.01 to 0.1) verified that activation by 9aa-Ins was more significantly impaired than repression (86% were no longer activated, and 65% were no longer repressed, P = 5.4 x 10 -6). Gene ontology analysis revealed that the 9aa insertion impaired GATA2-mediated activation of genes related to GPCR signaling and GATA2-mediated repression of genes related to innate immune machinery. The ectopically-regulated genes did not conform to a particular mechanism or pathway. Since it was unclear whether the transcriptional defects of the 9aa-Ins mutant reflect a unique inhibitory activity imparted by the 9aa sequence, we systematically varied the inter-zinc finger spacer length to establish whether any alterations can be tolerated. Using the genetic complementation assay, 2, 4, 6, 8, and 9aa spacer variants were compared with GATA2 for their capacity to regulate GATA2-activated genes (Hdc, Il1rl1, Gata1 and S1pr1) and repressed genes (Irf8, Il6st, Il6ra and Tifab). GATA2-mediated activation was compromised by insertions of two amino acids or more, whereas repression tolerated two and four amino acid insertions; 6, 8 and 9 were more inhibitory. Quantitative analyses revealed that a 6aa insertion reduced activation of the GATA2-activated genes by >50% of the wild type value, whereas the GATA2-repressed genes were still repressed by at least 50% (18% retention of activation and 83% retention of repression, P = 0.001). Thus, zinc finger spacing alterations differentially impacted activation versus repression. These results provide a rigorous foundation for interpreting variants that alter zinc finger spacing without disrupting vital finger residues. In vitro and in vivo functional analyses and molecular modeling are ongoing to further dissect the underlying mechanisms and ascertain the importance of genetic networks and circuits that are sensitive or resistant to human disease variants. Disclosures No relevant conflicts of interest to declare.

Analysis of the molecular evolution of histone variant H2A.Z using a linker-mediated complex strategy and yeast genetic complementation

The histone variant H2A.Z is deposited into chromatin by specific machinery and is required for genome functions. Using a linker-mediated complex strategy combined with yeast genetic complementation, we demonstrate evolutionary conservation of H2A.Z together with its chromatin incorporation and functions. This approach is applicable to the evolutionary analyses of proteins that form complexes with interactors.

The diadenylate cyclase, CdaA, is critical for Borrelia turicatae virulence and physiology

Relapsing fever (RF), caused by spirochetes of the genus Borrelia, is a globally distributed, vector-borne disease with high prevalence in developing countries. To date, signaling pathways required for infection and virulence of RF Borrelia spirochetes are unknown. Cyclic di-AMP (c-di-AMP), synthesized by diadenylate cyclases (DACs), is a second messenger predominantly found in Gram-positive organisms that is linked to virulence and essential physiological processes. Although Borrelia is Gram-negative, it encodes one DAC (CdaA), and its importance remains undefined. To investigate the contribution of c-di-AMP signaling in the RF bacterium, Borrelia turicatae, a cdaA mutant was generated. The mutant was significantly attenuated during murine infection, and genetic complementation reversed this phenotype. Because c-di-AMP is essential for viability in many bacteria, whole genome sequencing was performed on cdaA mutants, and single nucleotide polymorphisms identified potential suppressor mutations. Additionally, conditional mutation of cdaA confirmed that CdaA is important for normal growth and physiology. Interestingly, mutation of cdaA did not affect expression of homologs of virulence regulators whose levels are impacted by c-di-AMP signaling in the Lyme disease bacterium, Borrelia burgdorferi. Finally, the cdaA mutant had a significant growth defect when grown with salts, at decreased osmolarity, and without pyruvate. While the salt treatment phenotype was not reversed by genetic complementation, possibly due to suppressor mutations, growth defects at decreased osmolarity and in media lacking pyruvate could be attributed directly to cdaA inactivation. Overall, these results indicate CdaA is critical for B. turicatae pathogenesis and link c-di-AMP to osmoregulation and central metabolism in RF spirochetes.

The molecular clockwork of the suprachiasmatic nucleus is sufficient to co-ordinate phasing and stabilisation of sleep-wake cycles and enhance memory deficits in a clockless mouse

The timing and quality of sleep-wake cycles are regulated by interacting circadian and homeostatic mechanisms. Although the suprachiasmatic nucleus (SCN) is the principal circadian clock, local clocks are active across the brain and the respective sleep-regulatory roles of SCN and extra-SCN clocks are unclear. To determine the specific contribution(s) of the SCN, we used virally mediated genetic complementation, expressing Cryptochrome1 (Cry1) to restore circadian molecular competence to the SCN of globally clockless Cry1/Cry2-null mice. Under free-running conditions, the rest/activity behaviour of Cry1/Cry2-null controls which received EGFP (SCNCon) was arrhythmic, whereas Cry1-complemented mice (SCNCry1) had circadian behaviour comparable to that of Cry1,2-competent wild-types (WT). In SCNCon mice, sleep-wakefulness, assessed by electroencephalography/electromyography, also lacked circadian organisation. In SCNCry1 mice, however, it was comparable to WT, with consolidated vigilance states (wake, REM and NREM sleep) and rhythms in NREMS delta power and expression of REMS within total sleep. Wakefulness in SCNCon mice was more fragmented than in WT, with more wake-NREMS-wake transitions. This disruption was corrected in SCNCry1 mice. Following sleep deprivation, all mice showed an initial homeostatic increase in NREMS delta power. The SCNCon mice, however, had reduced, non-consolidated NREMS during the inactive phase of the recovery period. In contrast, the dynamics of homeostatic responses in the SCNCry1 mice were equivalent to WT. Finally, SCNCon mice exhibited poor sleep-dependent memory but this was corrected in SCNCry1mice. Therefore, the SCN clock is sufficient for circadian control of sleep-wake, facilitating initiation and maintenance of wake, promoting sleep consolidation, homeostatic dynamics, and sleep-dependent memory.Significance statementThe circadian timing system regulates sleep-wake cycles. The hypothalamic suprachiasmatic nucleus (SCN) is the principal circadian clock, but local clocks are also active across the brain and the respective roles of SCN and local clocks in regulating sleep are unclear. To determine, explicitly, the contribution of the SCN, we used virally mediated genetic complementation to restore SCN molecular circadian functions in otherwise genetically clockless mice. This initiated circadian activity-rest cycles, accompanied by circadian sleep-wake cycles, circadian patterning to the intensity of NREM sleep and circadian control of REM sleep as a proportion of total sleep. Consolidation of sleep-wake established normal dynamics of sleep homeostasis and enhanced sleep-dependent memory. Thus, the SCN is the principal and sufficient circadian regulator of sleep-wake.

Pseudozyma saprotrophic yeasts have retained a large effector arsenal, including functional Pep1 orthologs

SummaryThe basidiomycete smut fungi are predominantly plant parasitic, causing severe losses in some crops. Most species feature a saprotrophic haploid yeast stage, and several smut fungi are only known from this stage, with some isolated from habitats without suitable hosts, e.g. from Antarctica. Thus, these species are generally believed to be apathogenic, but recent findings that some of these might have a plant pathogenic sexual counterpart, casts doubts on the validity of this hypothesis. Here, four Pseudozyma genomes were re-annotated and compared to published smut pathogens and the well-characterised effector gene Pep1 from these species was checked for its ability to complement a Pep1 deletion strain of Ustilago maydis. It was found that 113 high-confidence putative effector proteins were conserved among smut and Pseudozyma genomes. Among these were several validated effector proteins, including Pep1. By genetic complementation we show that Pep1 homologs from the supposedly apathogenic yeasts restore virulence in Pep1-deficient mutants Ustilago maydis. Thus, it is concluded that Pseudozyma species have retained a suite of effectors. This hints at the possibility that Pseudozyma species have kept an unknown plant pathogenic stage for sexual recombination or that these effectors have positive effects when colonising plant surfaces.

Chlamydia trachomatis dapFEncodes a Bifunctional Enzyme Capable of Both D-Glutamate Racemase and Diaminopimelate Epimerase Activities

ABSTRACTPeptidoglycan is a sugar/amino acid polymer unique to bacteria and essential for division and cell shape maintenance. Thed-amino acids that make up its cross-linked stem peptides are not abundant in nature and must be synthesized by bacteriade novo.d-Glutamate is present at the second position of the pentapeptide stem and is strictly conserved in all bacterial species. In Gram-negative bacteria,d-glutamate is generated via the racemization ofl-glutamate by glutamate racemase (MurI).Chlamydia trachomatisis the leading cause of infectious blindness and sexually transmitted bacterial infections worldwide. While its genome encodes a majority of the enzymes involved in peptidoglycan synthesis, nomurIhomologue has ever been annotated. Recent studies have revealed the presence of peptidoglycan inC. trachomatisand confirmed that its pentapeptide includesd-glutamate. In this study, we show thatC. trachomatissynthesizesd-glutamate by utilizing a novel, bifunctional homologue of diaminopimelate epimerase (DapF). DapF catalyzes the final step in the synthesis ofmeso-diaminopimelate, another amino acid unique to peptidoglycan. Genetic complementation of anEscherichia coli murImutant demonstrated thatChlamydiaDapF can generated-glutamate. Biochemical analysis showed robust activity, but unlike canonical glutamate racemases, activity was dependent on the cofactor pyridoxal phosphate. Genetic complementation, enzymatic characterization, and bioinformatic analyses indicate that chlamydial DapF shares characteristics with other promiscuous/primordial enzymes, presenting a potential mechanism ford-glutamate synthesis not only inChlamydiabut also numerous other genera within thePlanctomycetes-Verrucomicrobiae-Chlamydiaesuperphylum that lack recognized glutamate racemases.IMPORTANCEHere we describe one of the last remaining “missing” steps in peptidoglycan synthesis in pathogenicChlamydiaspecies, the synthesis ofd-glutamate. We have determined that the diaminopimelate epimerase (DapF) encoded byChlamydia trachomatisis capable of carrying out both the epimerization of DAP and the pyridoxal phosphate-dependent racemization of glutamate. Enzyme promiscuity is thought to be the hallmark of early microbial life on this planet, and there is currently an active debate as to whether “moonlighting enzymes” represent primordial evolutionary relics or are a product of more recent reductionist evolutionary pressures. Given the large number ofChlamydiaspecies (as well as members of thePlanctomycetes-Verrucomicrobiae-Chlamydiaesuperphylum) that possess DapF but lack homologues of MurI, it is likely that DapF is a primordial isomerase that functions as both racemase and epimerase in these organisms, suggesting that specializedd-glutamate racemase enzymes never evolved in these microbes.