Chromatin remodeler Znhit1 controls bone morphogenetic protein signaling in embryonic lung tissue branching

Branching morphogenesis is a key process essential for lung and other organ development in which cellular and tissue architecture branch out to maximize surface area. While this process is known to be regulated by differential gene expression of ligands and receptors, how chromatin remodeling regulates this process remains unclear. Znhit1 (zinc finger HIT-type containing 1), acting as a chromatin remodeler, has previously been shown to control the deposition of the histone variant H2A.Z. Here, we demonstrate that Znhit1 also plays an important role in regulating lung branching. Using Znhit1 conditional KO mice, we show that Znhit1 deficiency in the embryonic lung epithelium leads to failure of branching morphogenesis and neonatal lethality, which is accompanied by reduced cell proliferation and increased cell apoptosis of the epithelium. The results from the transcriptome and the chromatin immunoprecipitation assay reveal that this is partially regulated by the derepression of Bmp4, encoding bone morphogenetic protein (BMP) 4, which is a direct target of H2A.Z. Furthermore, we show that inhibition of BMP signaling by the protein inhibitor Noggin rescues the lung branching defects of Znhit1 mutants ex vivo. Taken together, our study identifies the critical role of Znhit1/H2A.Z in embryonic lung morphogenesis via the regulation of BMP signaling.

Branching morphogenesis is a key process essential for lung and other organ development in which cellular and tissue architecture branch out to maximize surface area. While this process is known to be regulated by differential gene expression of ligands and receptors, how chromatin remodeling regulates this process remains unclear. Znhit1 (zinc finger HITtype containing 1), acting as a chromatin remodeler, has previously been shown to control the deposition of the histone variant H2A.Z. Here, we demonstrate that Znhit1 also plays an important role in regulating lung branching. Using Znhit1 conditional KO mice, we show that Znhit1 deficiency in the embryonic lung epithelium leads to failure of branching morphogenesis and neonatal lethality, which is accompanied by reduced cell proliferation and increased cell apoptosis of the epithelium. The results from the transcriptome and the chromatin immunoprecipitation assay reveal that this is partially regulated by the derepression of Bmp4, encoding bone morphogenetic protein (BMP) 4, which is a direct target of H2A.Z. Furthermore, we show that inhibition of BMP signaling by the protein inhibitor Noggin rescues the lung branching defects of Znhit1 mutants ex vivo. Taken together, our study identifies the critical role of Znhit1/H2A.Z in embryonic lung morphogenesis via the regulation of BMP signaling.
The lung is one of the essential organs that terrestrial vertebrates have evolved to adapt to life on land. The complex architecture of the lung enables the vital function of gas exchange, and lung development entails a series of remarkably elaborate branching events from the primary lung buds (1). In mice, the lung is derived from the ventral anterior foregut endoderm and appears at embryonic day 9 (E9) (2). The primary lung buds are formed at the embryonic stage E9.5-E12.5. During the subsequent pseudoglandular stage (E12.5-E16.5), the lung forms the primary airway pattern. SOX9-expressing proximal epithelial progenitors give rise to bronchioles and gas exchange units, and SOX2-expressing proximal regions gradually develop into the conductive airways in the mature lung. The functional adult lung is progressively formed after canalicular, saccular, and final alveolar stages.
The pattern formation of the lung is controlled by a series of important signaling molecules, including bone morphogenetic protein 4 (BMP4), SHH, fibroblast growth factor 10 (FGF10), and wingless (Wnt). Interactions between FGFs and BMPs are crucial to the formation of secondary lung buds. BMP4deficient mouse embryos die before E10, whereas the excessive activation of BMP4 antagonizes the FGF10-induced epithelial bud growth (3)(4)(5). These signaling molecules working together with their downstream target genes regulate lung morphological development. In addition, epigenetic regulations, including DNA methylation, histone modification, and chromatin remodeling, also play vital roles in the regulation of gene expression during lung development.
Chromatin remodeling is tightly coupled to lung branching. Loss of histone deacetylase 1/2 (5) or Sin3a (6), two components of the NuRD chromatin remodeling complex, impairs proximal airway epithelial differentiation partially through the activation of Bmp4 during mouse lung endoderm development. These findings suggest that chromatin remodeling is indispensable for proximal progenitor differentiation during lung development.
The replacement of canonical histone H2A with histone variant H2A.Z leads to chromatin remodeling and subsequent gene expression changes (7)(8)(9)(10)(11). As a key subunit of the SNF-2 related CBP activator protein complex, the zinc finger HITtype containing 1 (Znhit1) mediates the exchange of histone variant H2A.Z for H2A. Our recent studies have demonstrated that Znhit1 incorporates H2A.Z for transcriptional regulation of genes in Lgr5-positive stem cell maintenance (12), hematopoietic homeostasis (13), and meiotic initiation (14). However, the function of Znhit1/H2A.Z in mammalian pulmonary development remains unknown.
In this study, we show that Znhit1 deficiency in embryonic respiratory epithelial cells results in severe branching defects, accompanied by reduced proliferation and increased apoptosis of the entire lung epithelia. The deletion of Znhit1 leads to increased expression of Bmp4 in lung epithelium and overactivation of its downstream target SMAD; also the incorporation of H2A.Z in Bmp4 promoter region is Znhit1 dependent. Inhibition of BMP4 signaling in 3D lung epithelial organoids with the BMP antagonist Noggin can partially rescue the branching defects as shown by enhanced bud formation. Taken together, our work demonstrates that the epigenetic factor Znhit1 acts as a repressor of Bmp4 expression through H2A.Z in the embryonic epithelium during lung branching morphogenesis.

Znhit1 deletion impairs embryonic lung epithelium development
To determine the functions of Znhit1 in lung development, we first examined the expression pattern of Znhit1 during fetal lung development by immunochemistry histological staining. As shown in Fig. 1A, Znhit1 was widely expressed throughout the lung from E12.5 to E18.5 with nuclear localization.
To assess the potential role(s) of Znhit1 in epithelium development, we used Shh-Cre (15) to delete Znhit1 in the respiratory epithelium before birth. The efficiency of Cremediated epithelial deletion of Znhit1 was examined through quantitative RT-PCR (qRT-PCR) and immunostaining. The expression of Znhit1 was absent in the mutant lung epithelium at E12.5 but remained normal in the rest of the lung (Fig. 1B).
The survival of Shh-Cre; Znhit1 fl/fl (referred to as "Znhit1 cKO" [conditional KO] hereafter) mice during embryogenesis was as good as those of Znhit1 fl/fl and Shh-Cre; Znhit1 fl/+ (referred to as "control" hereafter) mice, with no significant weight/length changes (Figs. 1C; S1). While Znhit1 fl/fl and control mice both survived normally without any observable malformations, Znhit1-mutant neonatal mice died of respiratory failure immediately after birth. Further analysis of fetal lung development revealed that Znhit1 cKO embryos did form primary lung buds at E12.5 but exhibited multiple distal cysts instead of wellformed lung tissue by E18.5 (Figs. 1, D and E; S2), indicating an important function of Znhit1 in epithelial pattern formation.

Znhit1 is essential for embryonic lung distal epithelium branching
To determine whether Znhit1 regulates lung branching, we performed hematoxylin and eosin staining with lung sections of E12.5, E14.5, and E16.5. The observable branching defects at E14.5 developed into cystic morphology by E16.5 ( Fig. 2A). Along the proximodistal axis during branching morphogenesis, the lung endoderm with SOX2 expression marks the proximal airway progenitors (16,17), whereas SOX9 marks the distal progenitors (18,19). In E14.5 sections, the SOX2+ region aggressively extended and the SOX9+ region was restricted in the distal epithelial tips accompanied by branching morphogenesis in control mice. But the SOX9+ area showed an extended distal pattern and fewer branches in Znhit1-deficient epithelium while the patterning of the SOX2+ region remained largely unchanged (Fig. 2B).
The abnormal branching patterns of Znhit1 mutant mice suggest the possible functions of Znhit1 in regulating cell differentiation, proliferation, and cell death. Cell differentiation events during lung cell lineage specifications have been shown to regulate branching morphogenesis (20). To test whether the branching defects of Znhit1 mutant in the epithelium are due to its role in cell differentiation, we examined markers for major cell types of lung epithelium at E18.5. Airway club cell differentiation indicated by club cell secretory protein (CCSP), as well as AT1 cells and AT2 cells labeled by HOPX and proSPC staining, was undisturbed (Fig. 2C). The SOX2+ cells were lost in Znhit1-deficient lungs at E18.5 but remained unchanged in the esophagus (Fig. S3A), indicating a specific function of Znhit1 in lung epithelium development. The endothelial and smooth muscle cells also differentiated normally without Znhit1 (Fig. S3, B and C). These results suggest that Znhit1 in lung epithelium is dispensable for epithelial cell differentiation but exerts a regulatory function in lung branching.

Branching failure caused by Znhit1 deletion leads to decreased proliferation and increased apoptosis of lung epithelium
Previous studies have shown that Znhit1 plays a role in p53mediated cell apoptosis and cell cycle arrest in vitro (21,22). At the cellular level, the formation of new branches is also driven by the proliferation and cytoskeleton of lung epithelial cells. To examine whether cell proliferation is affected by the deletion of Znhit1, we performed immunofluorescence staining and quantification analyses. Phosphorylated histone H3 (PH3) is a marker of cell proliferation and labels cells in the G2 and M phases (23), whereas Ki67 can be found throughout the cell cycle except G0 phase (24,25). NKX2.1 was used to label fetal lung epithelial cells. Both cell number and proliferation of fetal lung epithelial and mesenchymal cells were analyzed and counted by costaining NKX2.1 and PH3 on the E14.5 fetal lung tissue sections. We found that the proliferation of epithelial cells was reduced in Znhit1 cKO lungs (p < 0.05) with unchanged cell polarity and structure (Figs. 3A; S4A). Loss of Znhit1 seemed to cause cell cycle arrest in lung epithelium as no significant differences were found in Ki67 staining (Fig. S4, B and C).
Next, we examined whether the level of cell apoptosis was changed in Znhit1-deficient lungs. By cleaved caspase-3 immunostaining, significantly increased apoptosis in epithelium was observed in Znhit1-deficient lungs (Fig. 3B). To test whether increased cell apoptosis affects lung branching, we dissected E12.5 lungs and performed an explant culture with pan-caspase inhibitor Z-VAD-FMK to inhibit apoptosis (Fig. S5A). Quantification of lung tips showed that lung branching is reduced in the Znhit1-deficient group, despite inhibition of apoptosis (Figs. 3C; S5B). These results suggest that reduced proliferation and increased apoptosis of lung epithelial cells induced by cKO of Znhit1 cannot explain branching defects of lung epithelium.

Znhit1 deficiency abolishes epithelium branching through activating BMP-SMADs signaling
To assess the molecular consequences of Znhit1 deficiency in early lung endoderm, we isolated epithelial cells from Znhit1 fl/fl and Znhit1 cKO lungs at E13.5 to perform RNA-Seq analysis. We found that 550 genes were significantly upregulated and 192 genes were significantly downregulated by Znhit1 deletion. Differently expressed genes were annotated to GO terms, including apoptosis, oxidative stress, and cell cyclerelated clustering (Fig. S6). Consistent with increased apoptosis of lung epithelial cells in the phenotypic analysis, we found increased expression of proapoptotic Bcl family genes BOK and Bax (p < 0.0001) and decreased expression of Bcl2L2 (p < 0.0001), which can inhibit the activity of Bax and promote cell survival (Fig. 4A). Moreover, Cdkn1a and Ccng1, related to cell cycle arrest, were also upregulated in the mutant epithelium, accounting for the cell proliferation disorder (Fig. 4A). Interestingly, Znhit1 deletion significantly upregulated the expression of Bmp4 in lung epithelium (Fig. 4A). Upregulation of Bmp4 expression in the epithelium is further confirmed by qRT-PCR (Fig. 4B). As reported previously (3,26), expression of Bmp4 is confined to the distal tips of the airway branches as observed in control lungs by in situ hybridization, but the expression of Bmp4 was elevated and pervaded throughout the epithelium of Znhit1 mutant lungs (Fig. 4C). Fgf10 signaling was not significantly altered in Znhit1 mutants (Fig. S7A), as were other critical genes involved in lung branching morphogenesis (Fig. S7B). To evaluate whether Bmp4 is directly regulated by H2A.Z, we performed anti-H2A.Z chromatin immunoprecipitation (ChIP) assay on embryonic lung samples. H2A.Z enrichment in the promoter region of Bmp4 significantly decreased in Znhit1-deficient lungs (Fig. 4D), supporting our hypothesis that Znhit1 promotes the incorporation of H2A.Z into the promoter region, thereby controlling Bmp4 transcription.
Canonical BMP-SMAD1/5/9 signaling is activated in Znhit1 cKO mice, and noggin treatment rescues lung budding formation in Znhit1 cKO lung epithelial organoids To examine the activation of BMP-SMAD1/5/9 signaling in Znhit1 cKO lungs, we used E13.5 lung sections to stain phosphor-SMAD1/5/9 (pSMAD1/5/9), a well-established indicator of BMP signaling activity (27). Expression of pSMAD1/ 5/9 exhibited an expanded pattern around the dilated epithelium with much stronger fluorescence intensity than that of controls (Fig. 5A), which is consistent with the expanded pattern of Bmp4. BMP signaling is an important regulator of anterior foregut endoderm development as well as a regulator of the proximal-distal patterning of the developing lung.
Previous studies have shown that activation of BMP signaling through epigenetic changes in epithelium inhibits lung branching and proximal lung development (5,28). Our data in lung explant culture did show a similar branching defect with BMP4 treatment (Fig. 5B). To determine whether the activation of BMP-SMAD1/5/9 signaling in the Znhit1deficient lungs contributes to branching defects described previously, we inhibited BMP signaling by using the BMP antagonist Noggin in an ex vivo lung organoid culture system. Lung epithelial organoids were generated from E12.5 lung epithelial cells in a 3D culture system, and the organoids' budding sites were identified by SOX2 and SOX9 immunofluorescent wholemount staining (Fig. 5C). Inhibition of BMP signaling with Noggin treatment improved budding formation of lung epithelial organoids, with loss of Znhit1 (p < 0.05). Consistent with SOX2+ cell loss and SOX9+ cell expansion in vivo, activation of BMP signaling also caused a decreased expression ratio of Sox2/Sox9, whereas BMP signaling inhibition increased the Sox2/Sox9 expression ratio significantly. The organoid culture from fetal lung epithelium demonstrated that the activation of intraepithelial Bmp4 caused by Znhit1 ablation hindered the formation of lung buds during branching development.

Discussion
One important issue in lung development is how epigenetic regulation is involved in cell fate determination during branching morphogenesis. Recent studies have shown that Znhit1 acts as a critical regulator of chromatin organization (12)(13)(14)29). In this study, we have provided convincing evidence that Znhit1 is essential for lung branching morphogenesis by coordinating with the histone variant H2A.Z to Znhit1 permits lung morphogenesis regulate Bmp4 expression (Fig. 6). Using Znhit1 cKO mice, we found the disrupted lung branching accompanied by decreased proliferation and increased apoptosis of lung epithelium in Znhit1 mutants. We demonstrate that Znhit1 can repress intraepithelial Bmp4 expression during embryonic development. Our results help to understand the mechanism by which lung development is tightly regulated under the context of chromatin.
The process of lung development, including specification, branching, and patterning, is fine tuned by key signaling pathways, such as the Wnt, BMP, and FGF pathways (30). Among these morphogen molecules, the moderate activity of BMP4 signaling plays a crucial role in regulating branching morphogenesis, and the derepression of Bmp4 expression in lung distal epithelium would lead to lung branching defects (5). Therefore, it is important to clarify the regulatory mechanism of Bmp4 transcription. Previous studies have shown that histone deacetylase 1/2-mediated histone deacetylation or Prmt5-mediated histone methylation is required for repressing Bmp4 transcription (5,28). Our work showed that excessive **p < 0.01 using unpaired two-tailed Student's t test. n = 5/group (without inhibitor), n = 4/group (with inhibitor). The scale bars represent 1000 μm. DAPI, 4 0 ,6-diamidino-2-phenylindole; Znhit1, zinc finger HIT-type containing 1. BMP signaling achieved by loss of Znhit1 in lung epithelium resulted in abnormal fetal lung formation, and inhibition of BMP signaling rescued budding formation in Znhit1 cKO lung epithelial organoids, partially. Compared with the control group, H2A.Z enrichment in the promoter region of Bmp4 decreased dramatically in Znhit1 cKO embryonic lungs (Fig. 4D), indicating that Znhit1-dependent H2A.Z enrichment in the promoter region directly controls Bmp4 transcription. Thus, we identify the essential role of the chromatin remodeler Znhit1 in branching morphogenesis by regulating Bmp4 expression.
Bmp4 is not only critical for lung development but also associated with chronic obstructive pulmonary disease (COPD) (31). Inhalational exposure to tobacco smoke is one of the major risk factors for COPD (32). And previous studies reported that the induced BMP4 signaling in human airway basal cells promotes cigarette smoking-related phenotypes (31). In consideration of the crucial role of Znhit1/H2A.Z in regulating Bmp4 expression, we speculate that Znhit1/H2A.Z may also get involved in the pathogenesis of COPD.

Znhit1 permits lung morphogenesis
H2A.Z incorporation into the promoter region during branching morphogenesis. Whether the regulatory mechanism of Znhit1/H2A.Z on BMP signaling is conserved during chronic injury and the repair process of airways needs to be addressed in further studies.

Experimental procedures
Mice Znhit1 fl/fl mice were generated by the Model Animal Research Center of Nanjing University (MARC) (12). Shh-Cre (The Jackson Laboratory, stock number 005622) mice were obtained from the Jackson Laboratory. For timed mating, embryonic day (E) 0.5 was determined by the presence of a copulation plug in the morning. Pregnant dams were sacrificed by CO 2 inhalation, and embryos were harvested at different embryonic stages. All strains were maintained in C57BL/6 background. Mice were housed in pathogen-free conditions according to the protocols approved by the Institutional Animal Care and Use Committee of Fudan University.

qRT-PCR
Total RNA was extracted with RNeasy Mini Kit (Qiagen), and the reverse transcription reaction was performed using GoScript Reverse Transcription System (Promega). qRT-PCRs were performed with GoTaq qPCR Master Mix (Promega) in triplicates on CFX96 Touch System (Bio-Rad). Primer pairs are listed in Table S1.

Isolation of epithelial cells by magnetic cell sorting
E13.5 lung lobes were harvested from control and Znhit1 cKO embryos to isolate epithelial cells. Dispase (Corning) was used for lobe digestion. After 15 min of digestion at 37 C, samples were transferred to 5 ml of Dulbecco's modified Eagle's medium (DMEM) containing 25 mM Hepes (Gibco) and 120 units of DNAse I (Sigma-Aldrich). Sample dissociation was performed on GentleMACS Dissociator (Miltenyi Biotec), and 40 μm nylon cell strainers (Corning) were used to get single cells. Cell suspensions that passed through the strainers were pelleted by centrifugation at 1500 rpm (433g) for 6 min at 4 C and resuspended in 10 ml DMEM (with Hepes and PenStrep) and centrifuged once again. Cells were resuspended in 90 μl of autoMACS Running Buffer (Miltenyi Biotec) and 10 μl FcR Blocking Reagent and incubated at 4 C for 10 min. Then, 10 μl of CD326 MicroBeads (Miltenyi Biotec) was added to bind epithelial cells. After incubation for 15 min at 4 C, cells were washed twice with autoMACS Running Buffer. Cell pellets were resuspended in 500 μl of Running Buffer to pass through a 40 μm nylon filter before sorting using an Auto-MACS Pro Separator (Miltenyi Biotec). CD326-positive (epithelial) and -negative (nonepithelial) cells were separated. Sorting efficiency was determined by the qRT-PCR of Epcam (epithelial) and Twist2 (mesenchymal).

RNA-Seq and analysis
RNA-Seq was performed on samples extracted from isolated lung epithelium at E13.5. The reverse transcription reaction was performed using the Ovation RNA-Seq System V2 kit (NuGEN). Sequencing and alignment were done by Berry Genomics. Znhit1 cKO and control samples (n = 2 each) were analyzed. The raw data were uniquely mapped to the mm10 genome by TopHat, version 1.4.1 (33). Expression values were assigned to gene level by Cufflinks, version 1.3.0 (34). All differentially expressed genes had a log2-transformed fold change >1.2 and a threshold of p < 0.05.

In situ hybridization
Mice embryos were washed with diethyl pyrocarbonatetreated PBS and fixed in 4% paraformaldehyde overnight at 4 C. Samples went through dehydration and paraffin embedding. In situ hybridization was performed on 6 μm paraffin sections with the RNA scope 2.0 kit (Advanced Cell Diagnostics). The probes used were as below: RNAscope Probe-Mm-Bmp4 (REF #401301).

ChIP-qPCR
Samples used for ChIP were freshly harvested embryonic lungs at E13.5 (n = 3). Samples were crosslinked with 1% formaldehyde for 10 min at room temperature, then quenched with glycine, and washed with PBS. ChIP protocol was done as previously described (12). The anti-H2A.Z antibody (ab4174; Abcam) and protein A/G magnetic beads (Millipore) were used for ChIP. ChIP-qPCR was performed with two pairs of designed primers in the promoter region of Bmp4 (Bmp4 #1 and Bmp4 #2 in Table S1).
Lung explant culture E11.5 lung buds were dissected from the embryos and then cultured as previously described (35) in the presence of either 250 ng/ml BMP4 (OrganRegen) or bovine serum albumin medium for 48 h.
Lung organoid culture E12.5 lungs were dissected from embryos and digested with prewarmed digestion buffer, containing collagenase I (Sigma) 200 U/ml and dispase II (Sigma) 6 U/ml for 15 min on a shaker at 37 C. The cells were centrifuged at 300g for 4 min and washed with wash medium (DMEM supplemented with 1% penicillin/streptomycin and 1% fetal bovine serum) and cold PBS. The harvested cells were collected and embedded in Matrigel (R&D Systems) and then seeded on a 48-well plate.

Statistical analysis
For qRT-PCR results and cell quantification, values are expressed as the mean ± SEM. Data were analyzed using a two-tailed unpaired Student's t test or one-way ANOVA test. p Values of 0.05 or less were considered statistically significant. Statistical analysis was performed with the GraphPad Prism 8 software (GraphPad Software, Inc).

Data availability
RNA-Seq RAW data have been deposited to the National Center for Biotechnology Information SRA database under accession number SRP325667. All other data described in the article are contained within the main text or supporting information.
Supporting information-This article contains supporting information.