SARS-CoV-2 disrupts host epigenetic regulation via histone mimicry.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged at the end of 2019 and caused the devastating global pandemic of coronavirus disease 2019 (COVID-19), in part because of its ability to effectively suppress host cell responses1-3. In rare cases, viral proteins dampen antiviral responses by mimicking critical regions of human histone proteins4-8, particularly those containing post-translational modifications required for transcriptional regulation9-11. Recent work has demonstrated that SARS-CoV-2 markedly disrupts host cell epigenetic regulation12-14. However, how SARS-CoV-2 controls the host cell epigenome and whether it uses histone mimicry to do so remain unclear. Here we show that the SARS-CoV-2 protein encoded by ORF8 (ORF8) functions as a histone mimic of the ARKS motifs in histone H3 to disrupt host cell epigenetic regulation. ORF8 is associated with chromatin, disrupts regulation of critical histone post-translational modifications and promotes chromatin compaction. Deletion of either the ORF8 gene or the histone mimic site attenuates the ability of SARS-CoV-2 to disrupt host cell chromatin, affects the transcriptional response to infection and attenuates viral genome copy number. These findings demonstrate a new function of ORF8 and a mechanism through which SARS-CoV-2 disrupts host cell epigenetic regulation. Further, this work provides a molecular basis for the finding that SARS-CoV-2 lacking ORF8 is associated with decreased severity of COVID-19.

Patterns of Ciliation and Ciliary Signaling in Cancer.

Among the factors that have been strongly implicated in regulating cancerous transformation, the primary monocilium (cilium) has gained increasing attention. The cilium is a small organelle extending from the plasma membrane, which provides a localized hub for concentration of transmembrane receptors. These receptors transmit signals from soluble factors (including Sonic hedgehog (SHH), platelet-derived growth factor (PDGF-AA), WNT, TGFß, NOTCH, and others) that regulate cell growth, as well as mechanosensory cues provided by flow or extracellular matrix. Ciliation is regulated by cell cycle, with most cells that are in G0 (quiescent) or early G1 ciliation and cilia typically absent in G2/M cells. Notably, while most cells organized in solid tissues are ciliated, cancerous transformation induces significant changes in ciliation. Most cancer cells lose cilia; medulloblastomas and basal cell carcinomas, dependent on an active SHH pathway, rely on ciliary maintenance. Changes in cancer cell ciliation are driven by core oncogenic pathways (EGFR, KRAS, AURKA, PI3K), and importantly ciliation status regulates functionality of those pathways. Ciliation is both influenced by targeted cancer therapies and linked to therapeutic resistance; recent studies suggest ciliation may also influence cancer cell metabolism and stem cell identity. We review recent studies defining the relationship between cilia and cancer.

Olfactory facilitation of visual categorization in the 4-month-old brain depends on visual demand.

To navigate their environment, infants rely on intersensory facilitation when unisensory perceptual demand is high, a principle known as inverse effectiveness. Given that this principle was mainly documented in the context of audiovisual stimulations, here we aim to determine whether it applies to olfactory-to-visual facilitation. We build on previous evidence that the mother's body odor facilitates face categorization in the 4-month-old brain, and investigate whether this effect depends on visual demand. Scalp electroencephalogram (EEG) was recorded in two groups of 4-month-old infants while they watched 6-Hz streams of visual stimuli with faces displayed every 6th stimulus to tag a face-selective response at 1 Hz. We used variable natural stimuli in one group (Nat Group), while stimuli were simplified in the other group (Simp Group) to reduce perceptual categorization demand. During visual stimulation, infants were alternatively exposed to their mother's versus a baseline odor. For both groups, we found an occipito-temporal face-selective response, but with a larger amplitude for the simplified stimuli, reflecting less demanding visual categorization. Importantly, the mother's body odor enhances the response to natural, but not to simplified, face stimuli, indicating that maternal odor improves face categorization when it is most demanding for the 4-month-old brain. Overall, this study demonstrates that the inverse effectiveness of intersensory facilitation applies to the sense of smell during early perceptual development. RESEARCH HIGHLIGHTS: Intersensory facilitation is a function of unisensory perceptual demand in infants (inverse effectiveness). This inverse relation between multisensory and unisensory perception has been mainly documented using audiovisual stimulations. Here we show that olfactory-to-visual facilitation depends on visual demand in 4-month-old infants. The inverse effectiveness of intersensory facilitation during early perceptual development applies to the sense of smell.

Olfactory-to-visual facilitation in the infant brain declines gradually from 4 to 12 months.

During infancy, intersensory facilitation declines gradually as unisensory perception develops. However, this trade-off was mainly investigated using audiovisual stimulations. Here, fifty 4- to 12-month-old infants (26 females, predominately White) were tested in 2017-2020 to determine whether the facilitating effect of their mother's body odor on neural face categorization, as previously observed at 4 months, decreases with age. In a baseline odor context, the results revealed a face-selective electroencephalographic response that increases and changes qualitatively between 4 and 12 months, marking improved face categorization. At the same time, the benefit of adding maternal odor fades with age (R2 = .31), indicating an inverse relation with the amplitude of the visual response, and generalizing to olfactory-visual interactions previous evidence from the audiovisual domain.

A Rare Coincidence of Three Inherited Diseases in a Family with Cardiomyopathy and Multiple Extracardiac Abnormalities.

A genetic diagnosis of primary cardiomyopathies can be a long-unmet need in patients with complex phenotypes. We investigated a three-generation family with cardiomyopathy and various extracardiac abnormalities that had long sought a precise diagnosis. The 41-year-old proband had hypertrophic cardiomyopathy (HCM), left ventricular noncompaction, myocardial fibrosis, arrhythmias, and a short stature. His sister showed HCM, myocardial hypertrabeculation and fibrosis, sensorineural deafness, and congenital genitourinary malformations. Their father had left ventricular hypertrophy (LVH). The proband's eldest daughter demonstrated developmental delay and seizures. We performed a clinical examination and whole-exome sequencing for all available family members. All patients with HCM/LVH shared a c.4411-2A>C variant in ALPK3, a recently known HCM-causative gene. Functional studies confirmed that this variant alters ALPK3 canonical splicing. Due to extracardiac symptoms in the female patients, we continued the search and found two additional single-gene disorders. The proband's sister had a p.Trp329Gly missense in GATA3, linked to hypoparathyroidism, sensorineural deafness, and renal dysplasia; his daughter had a p.Ser251del in WDR45, associated with beta-propeller protein-associated neurodegeneration. This unique case of three monogenic disorders in one family shows how a comprehensive approach with thorough phenotyping and extensive genetic testing of all symptomatic individuals provides precise diagnoses and appropriate follow-up, embodying the concept of personalized medicine. We also present the first example of a splicing functional study for ALPK3 and describe the genotype-phenotype correlations in cardiomyopathy.

Applicability of Diagnostic Criteria and High Prevalence of Familial Dysbetalipoproteinemia in Russia: A Pilot Study.

Familial dysbetalipoproteinemia (FD) is a highly atherogenic genetically based lipid disorder with an underestimated actual prevalence. In recent years, several biochemical algorithms have been developed to diagnose FD using available laboratory tests. The practical applicability of FD diagnostic criteria and the prevalence of FD in Russia have not been previously assessed. We demonstrated that the diagnostic algorithms of FD, including the diagnostic apoB levels, require correction, taking into account the distribution of apoB levels in the population. At the same time, a triglycerides cutoff ≥ 1.5 mmol/L may be a useful tool in identifying subjects with FD. In this study, a high prevalence of FD was detected: 0.67% (one in 150) based on the ε2ε2 haplotype and triglycerides levels ≥ 1.5 mmol/L. We also analyzed the presence and pathogenicity of APOE variants associated with autosomal dominant FD in a large research sample.

Histone methyltransferase activity programs nuclear peripheral genome positioning.

Spatial organization of the genome in the nucleus plays a critical role in development and regulation of transcription. A genomic region that resides at the nuclear periphery is part of the chromatin layer marked with histone H3 lysine 9 dimethyl (H3K9me2), but chromatin reorganization during cell differentiation can cause movement in and out of this nuclear compartment with patterns specific for individual cell fates. Here we describe a CRISPR-based system that allows visualization coupled with forced spatial relocalization of a target genomic locus in live cells. We demonstrate that a specified locus can be tethered to the nuclear periphery through direct binding to a dCas9-Lap2ß fusion protein at the nuclear membrane, or via targeting of a histone methyltransferase (HMT), G9a fused to dCas9, that promotes H3K9me2 labeling and localization to the nuclear periphery. The enzymatic activity of the HMT is sufficient to promote this repositioning, while disruption of the catalytic activity abolishes the localization effect. We further demonstrate that dCas9-G9a-mediated localization to the nuclear periphery is independent of nuclear actin polymerization. Our data suggest a function for epigenetic histone modifying enzymes in spatial chromatin organization and provide a system for tracking and labeling targeted genomic regions in live cells.

The Double Mutation DSG2-p.S363X and TBX20-p.D278X Is Associated with Left Ventricular Non-Compaction Cardiomyopathy: Case Report.

Left ventricular non-compaction cardiomyopathy (LVNC) is a rare heart disease, with or without left ventricular dysfunction, which is characterized by a two-layer structure of the myocardium and an increased number of trabeculae. The study of familial forms of LVNC is helpful for risk prediction and genetic counseling of relatives. Here, we present a family consisting of three members with LVNC. Using a next-generation sequencing approach a combination of two (likely) pathogenic nonsense mutations DSG2-p.S363X and TBX20-p.D278X was identified in all three patients. TBX20 encodes the cardiac T-box transcription factor 20. DSG2 encodes desmoglein-2, which is part of the cardiac desmosomes and belongs to the cadherin family. Since the identified nonsense variant (DSG2-p.S363X) is localized in the extracellular domain of DSG2, we performed in vitro cell transfection experiments. These experiments revealed the absence of truncated DSG2 at the plasma membrane, supporting the pathogenic relevance of DSG2-p.S363X. In conclusion, we suggest that in the future, these findings might be helpful for genetic screening and counseling of patients with LVNC.

Hemi- and Homozygous Loss-of-Function Mutations in DSG2 (Desmoglein-2) Cause Recessive Arrhythmogenic Cardiomyopathy with an Early Onset.

About 50% of patients with arrhythmogenic cardiomyopathy (ACM) carry a pathogenic or likely pathogenic mutation in the desmosomal genes. However, there is a significant number of patients without positive familial anamnesis. Therefore, the molecular reasons for ACM in these patients are frequently unknown and a genetic contribution might be underestimated. Here, we used a next-generation sequencing (NGS) approach and in addition single nucleotide polymor-phism (SNP) arrays for the genetic analysis of two independent index patients without familial medical history. Of note, this genetic strategy revealed a homozygous splice site mutation (DSG2-c.378+1G>T) in the first patient and a nonsense mutation (DSG2-p.L772X) in combination with a large deletion in DSG2 in the second one. In conclusion, a recessive inheritance pattern is likely for both cases, which might contribute to the hidden medical history in both families. This is the first report about these novel loss-of-function mutations in DSG2 that have not been previously identi-fied. Therefore, we suggest performing deep genetic analyses using NGS in combination with SNP arrays also for ACM index patients without obvious familial medical history. In the future, this finding might has relevance for the genetic counseling of similar cases.

Noncompaction cardiomyopathy is caused by a novel in-frame desmin (DES) deletion mutation within the 1A coiled-coil rod segment leading to a severe filament assembly defect.

Mutations in DES, encoding desmin protein, are associated with different kinds of skeletal and/or cardiac myopathies. However, it is unknown, whether DES mutations are associated with left ventricular hypertrabeculation (LVHT). Here, we performed a clinical examination and subsequent genetic analysis in a family, with two individuals presenting LVHT with conduction disease and skeletal myopathy. The genetic analysis revealed a novel small in-frame deletion within the DES gene, p.Q113_L115del, affecting the α-helical rod domain. Immunohistochemistry analysis of explanted myocardial tissue from the index patient revealed an abnormal cytoplasmic accumulation of desmin and a degraded sarcomeric structure. Cell transfection experiments with wild-type and mutant desmin verified the cytoplasmic aggregation and accumulation of mutant desmin. Cotransfection experiments were performed to model the heterozygous state of the patients and revealed a dominant negative effect of the mutant desmin on filament assembly. DES:p.Q113_L115del is classified as a pathogenic mutation associated with dilated cardiomyopathy with prominent LVHT.

Ganetespib limits ciliation and cystogenesis in autosomal-dominant polycystic kidney disease (ADPKD).

Autosomal-dominant polycystic kidney disease (ADPKD) is associated with progressive formation of renal cysts, kidney enlargement, hypertension, and typically end-stage renal disease. In ADPKD, inherited mutations disrupt function of the polycystins (encoded by PKD1 and PKD2), thus causing loss of a cyst-repressive signal emanating from the renal cilium. Genetic studies have suggested ciliary maintenance is essential for ADPKD pathogenesis. Heat shock protein 90 (HSP90) clients include multiple proteins linked to ciliary maintenance. We determined that ganetespib, a clinical HSP90 inhibitor, inhibited proteasomal repression of NEK8 and the Aurora-A activator trichoplein, rapidly activating Aurora-A kinase and causing ciliary loss in vitro. Using conditional mouse models for ADPKD, we performed long-term (10 or 50 wk) dosing experiments that demonstrated HSP90 inhibition caused durable in vivo loss of cilia, controlled cystic growth, and ameliorated symptoms induced by loss of Pkd1 or Pkd2. Ganetespib efficacy was not increased by combination with 2-deoxy-d-glucose, a glycolysis inhibitor showing some promise for ADPKD. These studies identify a new biologic activity for HSP90 and support a cilia-based mechanism for cyst repression.-Nikonova, A. S., Deneka, A. Y., Kiseleva, A. A., Korobeynikov, V., Gaponova, A., Serebriiskii, I. G., Kopp, M. C., Hensley, H. H., Seeger-Nukpezah, T. N., Somlo, S., Proia, D. A., Golemis, E. A. Ganetespib limits ciliation and cystogenesis in autosomal-dominant polycystic kidney disease (ADPKD).

Tandem repeats of Allium fistulosum associated with major chromosomal landmarks.

Tandem repeats are often associated with important chromosomal landmarks, such as centromeres, telomeres, subtelomeric, and other heterochromatic regions, and can be good candidates for molecular cytogenetic markers. Tandem repeats present in many plant species demonstrate dramatic differences in unit length, proportion in the genome, and chromosomal organization. Members of genus Allium with their large genomes represent a challenging task for current genetics. Using the next generation sequencing data, molecular, and cytogenetic methods, we discovered two tandemly organized repeats in the Allium fistulosum genome (2n = 2C = 16), HAT58 and CAT36. Together, these repeats comprise 0.25% of the bunching onion genome with 160,000 copies/1 C of HAT58 and 93,000 copies/1 C of CAT36. Fluorescent in situ hybridization (FISH) and C-banding showed that HAT58 and CAT36 associated with the interstitial and pericentromeric heterochromatin of the A. fistulosum chromosomes 5, 6, 7, and 8. FISH with HAT58 and CAT36 performed on A. cepa (2n = 2C = 16) and A. wakegi (2n = 2C = 16), a natural allodiploid hybrid between A. fistulosum and A. cepa, revealed that these repeats are species specific and produced specific hybridization patterns only on A. fistulosum chromosomes. Thus, the markers can be used in interspecific breeding programs for monitoring of alien genetic material. We applied Non-denaturing FISH that allowed detection of the repeat bearing chromosomes within 3 h. A polymorphism of the HAT58 chromosome location was observed. This finding suggests that the rapid evolution of the HAT58 repeat is still ongoing.

Voice categorization in the four-month-old human brain.

Voices are the most relevant social sounds for humans and therefore have crucial adaptive value in development. Neuroimaging studies in adults have demonstrated the existence of regions in the superior temporal sulcus that respond preferentially to voices. Yet, whether voices represent a functionally specific category in the young infant's mind is largely unknown. We developed a highly sensitive paradigm relying on fast periodic auditory stimulation (FPAS) combined with scalp electroencephalography (EEG) to demonstrate that the infant brain implements a reliable preferential response to voices early in life. Twenty-three 4-month-old infants listened to sequences containing non-vocal sounds from different categories presented at 3.33 Hz, with highly heterogeneous vocal sounds appearing every third stimulus (1.11 Hz). We were able to isolate a voice-selective response over temporal regions, and individual voice-selective responses were found in most infants within only a few minutes of stimulation. This selective response was significantly reduced for the same frequency-scrambled sounds, indicating that voice selectivity is not simply driven by the envelope and the spectral content of the sounds. Such a robust selective response to voices as early as 4 months of age suggests that the infant brain is endowed with the ability to rapidly develop a functional selectivity to this socially relevant category of sounds.

Growth factor-induced activation of MSK2 leads to phosphorylation of H3K9me2S10 and corresponding changes in gene expression.

Extracellular signals are transmitted through kinase cascades to modulate gene expression, but it remains unclear how epigenetic changes regulate this response. Here, we provide evidence that growth factor-stimulated changes in the transcript levels of many responsive genes are accompanied by increases in histone phosphorylation levels, specifically at histone H3 serine-10 when the adjacent lysine-9 is dimethylated (H3K9me2S10). Imaging and proteomic approaches show that epidermal growth factor (EGF) stimulation results in H3K9me2S10 phosphorylation, which occurs in genomic regions enriched for regulatory enhancers of EGF-responsive genes. We also demonstrate that the EGF-induced increase in H3K9me2S10ph is dependent on the nuclear kinase MSK2, and this subset of EGF-induced genes is dependent on MSK2 for transcription. Together, our work indicates that growth factor-induced changes in chromatin state can mediate the activation of downstream genes.

The secret life of chromatin tethers.

The nuclear envelope plays an essential role in organizing the genome inside of the nucleus. The inner nuclear membrane is coated with a meshwork of filamentous lamin proteins that provide a surface to organize a variety of cellular processes. A subset of nuclear lamina- and membrane-associated proteins functions as anchors to hold transcriptionally silent heterochromatin at the nuclear periphery. While most chromatin tethers are integral membrane proteins, a limited number are lamina-bound. One example is the mammalian proline-rich 14 (PRR14) protein. PRR14 is a recently characterized protein with unique function that is different from other known chromatin tethers. Here, we review our current understanding of PRR14 structure and function in organizing heterochromatin at the nuclear periphery.

PRR14 organizes H3K9me3-modified heterochromatin at the nuclear lamina.

The eukaryotic genome is organized in three dimensions within the nucleus. Transcriptionally active chromatin is spatially separated from silent heterochromatin, a large fraction of which is located at the nuclear periphery. However, the mechanisms by which chromatin is localized at the nuclear periphery remain poorly understood. Here we demonstrate that Proline Rich 14 (PRR14) protein organizes H3K9me3-modified heterochromatin at the nuclear lamina. We show that PRR14 dynamically associates with both the nuclear lamina and heterochromatin, and is able to reorganize heterochromatin in the nucleus of interphase cells independent of mitosis. We characterize two functional HP1-binding sites within PRR14 that contribute to its association with heterochromatin. We also demonstrate that PPR14 forms an anchoring surface for heterochromatin at the nuclear lamina where it interacts dynamically with HP1-associated chromatin. Our study proposes a model of dynamic heterochromatin organization at the nuclear lamina via the PRR14 tethering protein.

Loss of Pkd1 limits susceptibility to colitis and colorectal cancer.

Colorectal cancer (CRC) is one of the most common cancers, with an annual incidence of ~135,000 in the US, associated with ~50,000 deaths. Autosomal dominant polycystic kidney disease (ADPKD), associated with mutations disabling the PKD1 gene, affects as many as 1 in 1000. Intriguingly, some studies have suggested that individuals with germline mutations in PKD1 have reduced incidence of CRC, suggesting a genetic modifier function. Using mouse models, we here establish that loss of Pkd1 greatly reduces CRC incidence and tumor growth induced by loss of the tumor suppressor Apc. Growth of Pkd1-/-;Apc-/- organoids was reduced relative to Apc-/- organoids, indicating a cancer cell-intrinsic activity, even though Pkd1 loss enhanced activity of pro-oncogenic signaling pathways. Notably, Pkd1 loss increased colon barrier function, with Pkd1-deficient animals resistant to DSS-induced colitis, associated with upregulation of claudins that decrease permeability, and reduced T cell infiltration. Notably, Pkd1 loss caused greater sensitivity to activation of CFTR, a tumor suppressor in CRC, paralleling signaling relations in ADPKD. Overall, these data and other data suggest germline and somatic mutations in PKD1 may influence incidence, presentation, and treatment response in human CRC and other pathologies involving the colon.

Genetic landscape in Russian patients with familial left ventricular noncompaction.

Background: Left ventricular noncompaction (LVNC) cardiomyopathy is a disorder that can be complicated by heart failure, arrhythmias, thromboembolism, and sudden cardiac death. The aim of this study is to clarify the genetic landscape of LVNC in a large cohort of well-phenotyped Russian patients with LVNC, including 48 families (n=214). Methods: All index patients underwent clinical examination and genetic analysis, as well as family members who agreed to participate in the clinical study and/or in the genetic testing. The genetic testing included next generation sequencing and genetic classification according to ACMG guidelines. Results: A total of 55 alleles of 54 pathogenic and likely pathogenic variants in 24 genes were identified, with the largest number in the MYH7 and TTN genes. A significant proportion of variants -8 of 54 (14.8%) -have not been described earlier in other populations and may be specific to LVNC patients in Russia. In LVNC patients, the presence of each subsequent variant is associated with increased odds of having more severe LVNC subtypes than isolated LVNC with preserved ejection fraction. The corresponding odds ratio is 2.77 (1.37 -7.37; p <0.001) per variant after adjustment for sex, age, and family. Conclusion: Overall, the genetic analysis of LVNC patients, accompanied by cardiomyopathy-related family history analysis, resulted in a high diagnostic yield of 89.6%. These results suggest that genetic screening should be applied to the diagnosis and prognosis of LVNC patients.