SOX9: a stem cell transcriptional regulator of secreted niche signaling factors.

Hair follicles (HFs) undergo cyclical periods of growth, which are fueled by stem cells (SCs) at the base of the resting follicle. HF-SC formation occurs during HF development and requires transcription factor SOX9. Whether and how SOX9 functions in HF-SC maintenance remain unknown. By conditionally targeting Sox9 in adult HF-SCs, we show that SOX9 is essential for maintaining them. SOX9-deficient HF-SCs still transition from quiescence to proliferation and launch the subsequent hair cycle. However, once activated, bulge HF-SCs begin to differentiate into epidermal cells, which naturally lack SOX9. In addition, as HF-SC numbers dwindle, outer root sheath production is not sustained, and HF downgrowth arrests prematurely. Probing the mechanism, we used RNA sequencing (RNA-seq) to identify SOX9-dependent transcriptional changes and chromatin immunoprecipitation (ChIP) and deep sequencing (ChIP-seq) to identify SOX9-bound genes in HF-SCs. Intriguingly, a large cohort of SOX9-sensitive targets encode extracellular factors, most notably enhancers of Activin/pSMAD2 signaling. Moreover, compromising Activin signaling recapitulates SOX9-dependent defects, and Activin partially rescues them. Overall, our findings reveal roles for SOX9 in regulating adult HF-SC maintenance and suppressing epidermal differentiation in the niche. In addition, our studies expose a role for SCs in coordinating their own behavior in part through non-cell-autonomous signaling within the niche.

Pioneer factors govern super-enhancer dynamics in stem cell plasticity and lineage choice.

Adult stem cells occur in niches that balance self-renewal with lineage selection and progression during tissue homeostasis. Following injury, culture or transplantation, stem cells outside their niche often display fate flexibility. Here we show that super-enhancers underlie the identity, lineage commitment and plasticity of adult stem cells in vivo. Using hair follicle as a model, we map the global chromatin domains of hair follicle stem cells and their committed progenitors in their native microenvironments. We show that super-enhancers and their dense clusters ('epicentres') of transcription factor binding sites undergo remodelling upon lineage progression. New fate is acquired by decommissioning old and establishing new super-enhancers and/or epicentres, an auto-regulatory process that abates one master regulator subset while enhancing another. We further show that when outside their niche, either in vitro or in wound-repair, hair follicle stem cells dynamically remodel super-enhancers in response to changes in their microenvironment. Intriguingly, some key super-enhancers shift epicentres, enabling their genes to remain active and maintain a transitional state in an ever-changing transcriptional landscape. Finally, we identify SOX9 as a crucial chromatin rheostat of hair follicle stem cell super-enhancers, and provide functional evidence that super-enhancers are dynamic, dense transcription-factor-binding platforms which are acutely sensitive to pioneer master regulators whose levels define not only spatial and temporal features of lineage-status but also stemness, plasticity in transitional states and differentiation.

Engagement of the ATR-dependent DNA damage response at the human papillomavirus 18 replication centers during the initial amplification.

We have previously demonstrated that the human papillomavirus (HPV) genome replicates effectively in U2OS cells after transfection using electroporation. The transient extrachromosomal replication, stable maintenance, and late amplification of the viral genome could be studied for high- and low-risk mucosal and cutaneous papillomaviruses. Recent findings indicate that the cellular DNA damage response (DDR) is activated during the HPV life cycle and that the viral replication protein E1 might play a role in this process. We used a U2OS cell-based system to study E1-dependent DDR activation and the involvement of these pathways in viral transient replication. We demonstrated that the E1 protein could cause double-strand DNA breaks in the host genome by directly interacting with DNA. This activity leads to the induction of an ATM-dependent signaling cascade and cell cycle arrest in the S and G(2) phases. However, the transient replication of HPV genomes in U2OS cells induces the ATR-dependent pathway, as shown by the accumulation of γH2AX, ATR-interacting protein (ATRIP), and topoisomerase IIß-binding protein 1 (TopBP1) in viral replication centers. Viral oncogenes do not play a role in this activation, which is induced only through DNA replication or by replication proteins E1 and E2. The ATR pathway in viral replication centers is likely activated through DNA replication stress and might play an important role in engaging cellular DNA repair/recombination machinery for effective replication of the viral genome upon active amplification.

Bovine colostrum-derived antibodies against SARS-CoV-2 show great potential to serve as prophylactic agents.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to impose a serious burden on health systems globally. Despite worldwide vaccination, social distancing and wearing masks, the spread of the virus is ongoing. One of the mechanisms by which neutralizing antibodies (NAbs) block virus entry into cells encompasses interaction inhibition between the cell surface receptor angiotensin-converting enzyme 2 (ACE2) and the spike (S) protein of SARS-CoV-2. SARS-CoV-2-specific NAb development can be induced in the blood of cattle. Pregnant cows produce NAbs upon immunization, and antibodies move into the colostrum immediately before calving. Here, we immunized cows with SARS-CoV-2 S1 receptor binding domain (RBD) protein in proper adjuvant solutions, followed by one boost with SARS-CoV-2 trimeric S protein and purified immunoglobulins from colostrum. We demonstrate that this preparation indeed blocks the interaction between the trimeric S protein and ACE2 in different in vitro assays. Moreover, we describe the formulation of purified immunoglobulin preparation into a nasal spray. When administered to human subjects, the formulation persisted on the nasal mucosa for at least 4 hours, as determined by a clinical study. Therefore, we are presenting a solution that shows great potential to serve as a prophylactic agent against SARS-CoV-2 infection as an additional measure to vaccination and wearing masks. Moreover, our technology allows for rapid and versatile adaptation for preparing prophylactic treatments against other diseases using the defined characteristics of antibody movement into the colostrum.

Mechanism of genomic instability in cells infected with the high-risk human papillomaviruses.

In HPV-related cancers, the "high-risk" human papillomaviruses (HPVs) are frequently found integrated into the cellular genome. The integrated subgenomic HPV fragments express viral oncoproteins and carry an origin of DNA replication that is capable of initiating bidirectional DNA re-replication in the presence of HPV replication proteins E1 and E2, which ultimately leads to rearrangements within the locus of the integrated viral DNA. The current study indicates that the E1- and E2-dependent DNA replication from the integrated HPV origin follows the "onion skin"-type replication mode and generates a heterogeneous population of replication intermediates. These include linear, branched, open circular, and supercoiled plasmids, as identified by two-dimensional neutral-neutral gel-electrophoresis. We used immunofluorescence analysis to show that the DNA repair/recombination centers are assembled at the sites of the integrated HPV replication. These centers recruit viral and cellular replication proteins, the MRE complex, Ku70/80, ATM, Chk2, and, to some extent, ATRIP and Chk1 (S317). In addition, the synthesis of histone gammaH2AX, which is a hallmark of DNA double strand breaks, is induced, and Chk2 is activated by phosphorylation in the HPV-replicating cells. These changes suggest that the integrated HPV replication intermediates are processed by the activated cellular DNA repair/recombination machinery, which results in cross-chromosomal translocations as detected by metaphase FISH. We also confirmed that the replicating HPV episomes that expressed the physiological levels of viral replication proteins could induce genomic instability in the cells with integrated HPV. We conclude that the HPV replication origin within the host chromosome is one of the key factors that triggers the development of HPV-associated cancers. It could be used as a starting point for the "onion skin"-type of DNA replication whenever the HPV plasmid exists in the same cell, which endangers the host genomic integrity during the initial integration and after the de novo infection.

Two separate replication modes of the bovine papillomavirus BPV1 origin of replication that have different sensitivity to p53.

We have shown previously that transient amplificational replication of reporter plasmids that carry the papillomavirus origin of replication is efficiently blocked by p53 protein in several cell lines. We demonstrate now that the replication of stably maintained episomal bovine papillomavirus BPV1 URR (upstream regulatory region) reporter plasmid is not sensitive to p53. In addition, these two replication modes--initial transient amplificational replication and stable maintenance replication of essentially the same BPV1 URR reporter plasmid--can take place in the same cells, where amplificational replication does not interfere with the stable maintenance replication. These data suggest that BPV1 replicons could follow two clearly separable replication mechanisms during initial amplification and during stable extrachromosomal maintenance.

Papillomavirus DNA replication - from initiation to genomic instability.

Papillomaviruses establish their productive life cycle in stratified epithelium or mucosa, where the undifferentiated proliferating keratinocytes are the initial targets for the productive viral infection. Papillomaviruses have evolved mechanisms to adapt to the normal cellular growth control pathways and to adjust their DNA replication and maintenance cycle to contend with the cellular differentiation. We provide overview of the papillomavirus DNA replication in the differentiating epithelium and describe the molecular interactions important for viral DNA replication on all steps of the viral life cycle.

Genomic instability of the host cell induced by the human papillomavirus replication machinery.

Development of invasive cervical cancer upon infection by 'high-risk' human papillomavirus (HPV) in humans is a stepwise process in which some of the initially episomal 'high-risk' type of HPVs (HR-HPVs) integrate randomly into the host cell genome. We show that HPV replication proteins E1 and E2 are capable of inducing overamplification of the genomic locus where HPV origin has been integrated. Clonal analysis of the cells in which the replication from integrated HPV origin was induced showed excision, rearrangement and de novo integration of the HPV containing and flanking cellular sequences. These data suggest that papillomavirus replication machinery is capable of inducing genomic changes of the host cell that may facilitate the formation of the HPV-dependent cancer cell.