RESUMEN
The transcription factor PU.1 occupies a central role in controlling myeloid and early B-cell development, and its correct lineage-specific expression is critical for the differentiation choice of hematopoietic progenitors. However, little is known of how this tissue-specific pattern is established. We previously identified an upstream regulatory cis element whose targeted deletion in mice decreases PU.1 expression and causes leukemia. We show here that the upstream regulatory cis element alone is insufficient to confer physiologic PU.1 expression in mice but requires the cooperation with other, previously unidentified elements. Using a combination of transgenic studies, global chromatin assays, and detailed molecular analyses we present evidence that PU.1 is regulated by a novel mechanism involving cross talk between different cis elements together with lineage-restricted autoregulation. In this model, PU.1 regulates its expression in B cells and macrophages by differentially associating with cell type-specific transcription factors at one of its cis-regulatory elements to establish differential activity patterns at other elements.
Asunto(s)
Linfocitos B/metabolismo , Regulación de la Expresión Génica/genética , Células Mieloides/metabolismo , Proteínas Proto-Oncogénicas/genética , Elementos Reguladores de la Transcripción/genética , Transactivadores/genética , Animales , Southern Blotting , Western Blotting , Separación Celular , Retroalimentación Fisiológica/fisiología , Citometría de Flujo , Expresión Génica , Hematopoyesis/genética , Humanos , Ratones , Ratones Transgénicos , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Proto-Oncogénicas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transactivadores/metabolismoRESUMEN
A single-chain Fv (scFv) fragment derived from the murine antibody 4G7, specific for human lymphocyte CD19, was engineered for stability and expression in Escherichia coli in view of future use as a therapeutic protein. We compared two orthogonal knowledge-based procedures. In one approach, we designed a mutant with 14 single amino-acid substitutions predicted to correct destabilizing residues in the 4G7-wt sequence to create 4G7-mut. In the second variant, the murine CDRs were grafted to the human acceptor framework huVkappa3-huV(H)3, with 11 additional point mutations introduced to obtain a better match between CDR graft and acceptor framework, to arrive at 4G7-graft. Compared to 4G7-wt, 4G7-mut showed greater thermodynamic stability in guanidinium chloride-induced equilibrium denaturation experiments and somewhat greater stability in human serum. The loop graft maintained the comparatively high stability of the murine loop donor, but did not improve it further. Our analysis indicates that this is due to subtle strain introduced between CDRs and framework, mitigating the otherwise highly favorable properties of the human acceptor framework. This slight strain in the loop graft is also reflected in the binding affinities for CD19 on leukemic cells of 8.4 nM for 4G7-wt, 16.4 nM for 4G7-mut and 30.0 nM for 4G7-graft. This comparison of knowledge-based mutation and loop-grafting-based approaches will be important, when moving molecules forward to therapeutic applications.
Asunto(s)
Antígenos CD19/genética , Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Mutación Puntual/genética , Proteínas Recombinantes de Fusión/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Animales , Afinidad de Anticuerpos/genética , Antígenos CD19/química , Antígenos CD19/metabolismo , Cromatografía en Gel , Escherichia coli/genética , Ingeniería Genética , Humanos , Cadenas Pesadas de Inmunoglobulina/química , Cadenas Pesadas de Inmunoglobulina/metabolismo , Región Variable de Inmunoglobulina/química , Región Variable de Inmunoglobulina/metabolismo , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Desnaturalización Proteica/genética , Estabilidad Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Solubilidad , TermodinámicaRESUMEN
DNA methylation is a dynamic epigenetic mark that undergoes extensive changes during differentiation of self-renewing stem cells. However, whether these changes are the cause or consequence of stem cell fate remains unknown. Here, we show that alternative functional programs of hematopoietic stem cells (HSCs) are governed by gradual differences in methylation levels. Constitutive methylation is essential for HSC self-renewal but dispensable for homing, cell cycle control and suppression of apoptosis. Notably, HSCs from mice with reduced DNA methyltransferase 1 activity cannot suppress key myeloerythroid regulators and thus can differentiate into myeloerythroid, but not lymphoid, progeny. A similar methylation dosage effect controls stem cell function in leukemia. These data identify DNA methylation as an essential epigenetic mechanism to protect stem cells from premature activation of predominant differentiation programs and suggest that methylation dynamics determine stem cell functions in tissue homeostasis and cancer.