Regulatory gene circuits with positive feedback loops control stem cell differentiation, but several mechanisms can contribute to positive feedback. Here, we dissect feedback mechanisms through which the transcription factor PU.1 controls lymphoid and myeloid differentiation. Quantitative live-cell imaging revealed that developing B-cells decrease PU.1 levels by reducing PU.1 transcription, whereas developing macrophages increase PU.1 levels by lengthening their cell cycles, which causes stable PU.1 accumulation. Exogenous PU.1 expression in progenitors increases endogenous PU.1 levels by inducing cell-cycle lengthening, implying positive feedback between a regulatory factor and the cell cycle. Mathematical modeling showed that this cell-cycle coupled feedback architecture effectively stabilizes a slow-dividing differentiated state. These results show that cell cycle duration functions as an integral part of a positive auto-regulatory circuit to control cell fate.

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Highlights d T cell-induced IFN-g correlates the best to immune checkpoint blockade (ICB) therapy d Immune signatures increase in on-therapy patients regardless of response to therapy d ICB therapy decreases MYC and WNT signaling in patients with clinical response d IFN-g exposure of melanoma cells leads to a conserved transcriptome signature

Although dependent on the integrity of a central pacemaker in the suprachiasmatic nucleus of the hypothalamus (SCN), endogenous daily (circadian) rhythms are expressed in a wide variety of peripheral organs. The pathways by which the pacemaker controls the periphery are unclear. Here, we used parabiosis between intact and SCN-lesioned mice to show that nonneural (behavioral or bloodborne) signals are adequate to maintain circadian rhythms of clock gene expression in liver and kidney, but not in heart, spleen, or skeletal muscle. These results indicate that the SCN regulates expression of circadian oscillations in different peripheral organs by diverse pathways.A wide variety of physiological events and behaviors exhibit pronounced endogenous daily (circadian) rhythms. Experimental destruction of the suprachiasmatic nucleus of the hypothalamus (SCN) results in arrhythmicity that is reversed by neurotransplantation of this structure. Persistence of rhythmicity depends on the operation of transcriptional-translational feedback loops among the products of critical genes, including Per1, Per2, and Bmal1 within the SCN. Circadian oscillations of Per1, Per2, and Bmal1 also occur in a variety of peripheral organs. Evidence has accumulated for both neural and humoral control of peripheral rhythms. For example, serum shock initiates oscillations of mPer1 expression in cultured hepatocytes and HeLa cells (1), and implants of fibroblasts that receive no innervation adopt the circadian phase of the host (2). Endocrine signals, including glucocorticoids, angiotensin II, and retinoic acid can shift the phase of peripheral clock gene expression (3-5). In addition, metabolites such as glucose may directly entrain peripheral oscillators or act indirectly to induce endocrine signals that regulate circadian rhythms of gene expression (6). On the other hand, the autonomic innervation of peripheral organs provides a potential pathway for entrainment (7-9). For example, SCN lesions that compromise catecholamine rhythms eliminate oscillations of clock gene expression in mouse liver (10).Neural and endocrine pathways for peripheral entrainment are not mutually exclusive. Furthermore, different organs may vary in their dependence on one or another entraining signal. Such variation is not without precedent. Whereas behavioral rhythms appear to depend on humoral outputs of the SCN (11), endocrine rhythms may rely on axonal projections (12).The technique of parabiosis offers unique advantages for investigation of the importance of blood-borne cues in the control of a variety of physiologic systems. This approach has been exploited in the study of cockroach circadian rhythms (13). Despite its useful application to study of metabolic signals in mice (14, 15), the effect on peripheral circadian rhythms of establishing vascular exchange without neural communication has not previously been investigated in vertebrates. We now report that parabiotic linkage of SCN-lesioned mice to intact partners reinstates circadian rhythmicity in some, but not o...

GATA-3 expression is crucial for T cell development and peaks during commitment to the T-cell lineage, midway through the CD4−CD8− (DN) 1-3 stages. We used RNA interference and conditional deletion to reduce GATA-3 protein acutely at specific points during T-cell differentiation in vitro. Even moderate GATA-3 reduction killed DN1 cells, delayed progression to DN2 stage, skewed DN2 gene regulation, and blocked appearance of DN3 phenotype. Although a Bcl-2 transgene rescued DN1 survival and improved DN2 cell generation, it did not restore DN3 differentiation. Gene expression analyses (qPCR, RNA-seq) showed that GATA-3-deficient DN2 cells quickly upregulated genes including Spi1 (PU.1) and Bcl11a and downregulated genes including Cpa3, Ets1, Zfpm1, Bcl11b, Il9r and Il17rb, with gene-specific kinetics and dose-dependencies. These targets could mediate two distinct roles played by GATA-3 in lineage commitment, as revealed by removing wildtype or GATA-3-deficient early T-lineage cells from environmental Notch signals. GATA-3 worked as a potent repressor of B-cell potential even at low expression levels, so that only full deletion of GATA-3 enabled pro-T cells to reveal B-cell potential. The ability of GATA-3 to block B-cell development did not require T-lineage commitment factor Bcl11b. In prethymic multipotent precursors, however, titration of GATA-3 activity using tamoxifen-inducible GATA-3 showed that GATA-3 inhibits B and myeloid developmental alternatives at different threshold doses. Furthermore, differential impacts of a GATA-3 obligate repressor construct imply that B and myeloid development are inhibited through distinct transcriptional mechanisms. Thus, the pattern of GATA-3 expression sequentially produces B-lineage exclusion, T-lineage progression, and myeloid-lineage exclusion for commitment.