Intestinal epithelial homeostasis is maintained by adult intestinal stem cells, which, alongside Paneth cells, appear after birth in the neonatal period. We aimed to identify regulators of neonatal intestinal epithelial development by testing a small library of epigenetic modifier inhibitors in Paneth cell–skewed organoid cultures. We found that lysine-specific demethylase 1A (Kdm1a/Lsd1) is absolutely required for Paneth cell differentiation. Lsd1-deficient crypts, devoid of Paneth cells, are still able to form organoids without a requirement of exogenous or endogenous Wnt. Mechanistically, we find that LSD1 enzymatically represses genes that are normally expressed only in fetal and neonatal epithelium. This gene profile is similar to what is seen in repairing epithelium, and we find that Lsd1-deficient epithelium has superior regenerative capacities after irradiation injury. In summary, we found an important regulator of neonatal intestinal development and identified a druggable target to reprogram intestinal epithelium toward a reparative state.
30Intestinal epithelial homeostasis is maintained by adult intestinal stem cells, which, alongside 31 Paneth cells, appear after birth in the neonatal period. We aimed to identify new regulators of 32 neonatal intestinal epithelial development by testing a small library of epigenetic modifier 33 inhibitors in Paneth cell-skewed organoid cultures. We found that Lysine-specific 34 demethylase 1A (Kdm1a/Lsd1) is absolutely required for Paneth cell differentiation. Lsd1-35 deficient crypts, devoid of Paneth cells, are still able to form organoids without a requirement 36 of exogenous or endogenous Wnt. Mechanistically, we find that LSD1 represses genes that 37 are normally expressed in fetal and neonatal epithelium. This gene profile is similar to what is 38 seen in repairing epithelium, and indeed, we find that Lsd1-deficient epithelium has superior 39 regenerative capacities after irradiation injury. In summary, we found an important regulator 40 of neonatal intestinal development and identified a druggable target to reprogram intestinal 41 epithelium towards a reparative state. 42indicate PC + crypts based on presence of granular cells, asterisks indicate PCcrypts. D, Brightfield images and
Infectious and inflammatory diseases in the intestine remain a serious threat for patients world-wide. Reprogramming of the intestinal epithelium towards a protective effector state is important to manage inflammation and immunity and can be therapeutically targeted. The role of epigenetic regulatory enzymes within these processes is not yet defined. Here, we use a mouse model that has an intestinal-epithelial specific deletion of the histone demethylase Lsd1 (cKO mice), which maintains the epithelium in a fixed reparative state. Challenge of cKO mice with bacteria-induced colitis or a helminth infection model both resulted in increased pathogenesis. Mechanistically, we discovered that LSD1 is important for goblet cell maturation and goblet-cell effector molecules such as RELMß. We propose that this may be in part mediated by directly controlling genes that facilitate cytoskeletal organization, which is important in goblet cell biology. This study therefore identifies intestinal-epithelial epigenetic regulation by LSD1 as a critical element in host protection from infection.
Chromatin immunoprecipitation (ChIP) enables mapping of specific histone modifications or chromatin‐associated factors in the genome and represents a powerful tool in the study of chromatin and genome regulation. Importantly, recent technological advances that couple ChIP with whole‐genome high‐throughput sequencing (ChIP‐seq) now allow the mapping of chromatin factors throughout the genome. However, the requirement for large amounts of ChIP‐seq input material has long made it challenging to assess chromatin profiles of cell types only available in limited numbers. For many cell types, it is not feasible to reach high numbers when collecting them as homogeneous cell populations in vivo. Nonetheless, it is an advantage to work with pure cell populations to reach robust biological conclusions. Here, we review (a) how ChIP protocols have been scaled down for use with as little as a few hundred cells; (b) which considerations to be aware of when preparing small‐scale ChIP‐seq and analyzing data; and (c) the potential of small‐scale ChIP‐seq datasets for elucidating chromatin dynamics in various biological systems, including some examples such as oocyte maturation and preimplantation embryo development. This article is categorized under: Laboratory Methods and Technologies > Genetic/Genomic Methods Developmental Biology > Developmental Processes in Health and Disease Biological Mechanisms > Cell Fates
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