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Principal Investigator: Lothar Hennighausen
Organization: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
Fiscal Year: 2021
Award: $337,442
Funding agency: National Institute of Diabetes and Digestive and Kidney Diseases
Objective 1
During this reporting period the Laboratory of Genetics and Physiology has made major progress in deciphering and understanding molecular mechanisms underlying hormone-controlled genetic programs in the mammary gland (PMID: 33854063).
Enhancers are transcription component platforms that control the location, timing and intensity of gene expression. While current approaches, such as the ChIP-seq and physical contact studies, are useful in identifying candidate enhancers, their biological predictions are limited and validation through genetic experiments is needed. Enhancers are occupied by multiple transcription factors (TFs) that might bind directly to DNA through their respective recognition motifs or indirectly through tethering. Since experimental genetic studies generally ablate the entire enhancer, the structural and functional contribution of individual TFs remains to be understood.
Several hundred genes are uniquely expressed in mammary tissue and activated by pregnancy and lactation hormones through the tyrosine kinase JAK2 and the transcription factor Signal Transducer and Activator of Transcription (STAT) 5. STAT5 is activated by prolactin and it controls mammary alveolar development during pregnancy and the activation of genetic programs resulting in lactation. While most STAT5 target genes are highly induced during pregnancy and to a lesser extent during lactation, the activation of the Csn1s2b gene occurs preferentially during lactation, possibly through enhancers that are specifically established after parturition. ChIP-seq profiles for STAT5 and H3K27ac and other mammary-enriched TFs suggested the presence of highly complex mammary enhancers. Although most of these enhancers appear to depend on STAT5 as the anchor for the establishment of larger protein complexes, the stage-specific establishment of enhancers remains to be understood. It is not known why seemingly structurally identical enhancers can be activated by pregnancy hormones either during pregnancy or lactation.
Here we have investigated enhancers that activate gene expression several hundred-fold exclusively in the lactating mouse mammary gland. Using ChIP-seq for activating histone marks and transcription factors, we identified two candidate enhancers and one super-enhancers in the Csn1s2b locus. Through experimental mouse genetics, we dissected the lactation-specific distal enhancer bound by the mammary-enriched transcription factors STAT5 and NFIB and the glucocorticoid receptor. While deletions of canonical binding motifs for NFIB and STAT5, individually or combined, have a limited biological impact, a non-canonical STAT5 site is essential for enhancer activity during lactation. In contrast, the intronic enhancer contributes to gene expression only in late pregnancy and early lactation, possibly by interacting with the distal enhancer. A downstream super-enhancer, which physically interacts with the distal enhancer, is required for the functional establishment of the Csn1s2b promoter and gene activation. Lastly, NFIB binding in the promoter region fine-tunes Csn1s2b expression.
Our study provides comprehensive insight into the anatomy and biology of regulatory elements that employ the JAK/STAT signaling pathway and preferentially activate gene expression during lactation.
Objective 2
SARS-CoV-2 infection of human airway epithelium activates genetic programs leading to progressive hyperinflammation in COVID-19 patients. Although SARS-CoV-2 infection of lung epithelium is a critical driver of disease, extrapulmonary manifestations of coronavirus disease 2019 (COVID-19) infection have been associated with direct viral damage of several tissues, including the kidney. The angiotensin-converting enzyme 2 (ACE2) receptor is the gateway for SARS-CoV-2 into cells and deciphering its regulation is relevant in understanding the viral damage and COVID-19 pathology. SARS-CoV-2 RNA has also been detected in milk, suggesting the possibility of vertical viral transmission and the need for an understanding of the regulation of the ACE2 receptor in mammary tissue during pregnancy and lactation.
Interferons regulate novel ACE2 isoforms in human lung and kidney primary cells
SARS-CoV-2 infection of bronchial cells triggers cytokine storms that can cause widespread pathology in a range of tissues. In addition, viral infection of the kidney contributes to organ damage, leading to increased mortality, especially in patients with previously diagnosed renal insufficiency.
Using genome-scale experiments we have identified the entire set of genes (transcriptomes) activated in human primary airway cells by interferons and we deciphered underlying regulatory elements controlling their expression (PMID: 34079039). Deciphering the regulation of the angiotensin-converting enzyme 2 (ACE2), the receptor for SARS-CoV-2, is paramount for understanding the cell tropism of SARS-CoV-2 infection. Using ChIP-seq for activating histone marks and Pol II loading, we identified candidate enhancer elements controlling the ACE2 locus, including the novel intronic dACE2 promoter. Employing RNA-seq, we demonstrate that interferons activate expression of dACE and, to a lesser extent, the genuine ACE2 gene. Interferon-induced gene expression was mitigated by the JAK inhibitors baricitinib and ruxolitinib used therapeutically in COVID-19 patients. Through integrating RNA-seq and ChIP-seq data we provide an in-depth understanding of genetic programs activated by interferons, and our study highlights JAK inhibitors as suitable tools to suppress these in bronchial cells.
SARS-CoV-2 infection of kidney leads to increased mortality and we conducted experiments to identify the genetic programs activated in Human Primary Proximal Tubule (HPPT) cells by interferons and their suppression by ruxolitinib (PMID: 34345808). Integration of our data with those form acute kidney injury and COVID-19 patients, as well as other tissues, permitted the identification of kidney-specific interferon responses. We also characterized the recently discovered dACE2 isoform and identified candidate interferon-activated enhancers controlling the extended ACE2 locus.
Taken together, our studies provide an in-depth understanding of genetic programs activated in lung and kidney and the dampening role of JAK inhibitors, which has practical implications for their use in treating COVID-19 patients.
ACE2 expression in mammary tissue during pregnancy and lactation
Viral RNA was detected in breast milk, raising the possibility of vertical transmission. To understand molecular aspects of viral entry into breast cells, we used mice as a proxy and investigated Ace2 regulation in the mammary gland during pregnancy and lactation (PMID: 32966801). We demonstrated that Ace2 expression in mammary tissue is induced during pregnancy and lactation, which coincides with the activation of intronic enhancers. These enhancers are occupied by the prolactin-activated transcription factor STAT5 and additional regulatory factors, including RNA polymerase II. Deletion of Stat5a results in decommissioning of the enhancers and a more than 80% reduction of Ace2 mRNA. JAK/STAT components are present in a range of SARS-CoV-2 target cells, opening the possibility that cytokines contribute to the viral load and extrapulmonary pathophysiology.
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