A large fraction of the mammalian genome is organized into inactive chromosomal domains along the nuclear lamina. The mechanism by which these lamina associated domains (LADs) are established remains to be elucidated. Using genomic repositioning assays, we show that LADs, spanning the developmentally regulated IgH and Cyp3a loci contain discrete DNA regions that associate chromatin with the nuclear lamina and repress gene activity in fibroblasts. Lamina interaction is established during mitosis and likely involves the localized recruitment of Lamin B during late anaphase. Fine-scale mapping of LADs reveals numerous lamina-associating sequences (LASs), which are enriched for a GAGA motif. This repeated motif directs lamina association and is bound by the transcriptional repressor cKrox, in a complex with HDAC3 and Lap2β. Knockdown of cKrox or HDAC3 results in dissociation of LASs/LADs from the nuclear lamina. These results reveal a mechanism that couples nuclear compartmentalization of chromatin domains with the control of gene activity.

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The transcription factor Ikaros is essential for B cell development. However, its molecular functions in B cell fate specification and commitment have remained elusive. We show here that the transcription factor EBF restored the generation of CD19(+) pro-B cells from Ikaros-deficient hematopoietic progenitors. Notably, these pro-B cells, despite having normal expression of the transcription factors EBF and Pax5, were not committed to the B cell fate. They also failed to recombine variable gene segments at the immunoglobulin heavy-chain locus. Ikaros promoted heavy-chain gene rearrangements by inducing expression of the recombination-activating genes as well as by controlling accessibility of the variable gene segments and compaction of the immunoglobulin heavy-chain locus. Thus, Ikaros is an obligate component of a network that regulates B cell fate commitment and immunoglobulin heavy-chain gene recombination.

Mammalian sperm are incapable of fertilizing eggs immediately after ejaculation; they acquire fertilization capacity after residing in the female tract for a finite period of time. The physiological changes sperm undergo in the female reproductive tract that render sperm able to fertilize constitute the phenomenon of "sperm capacitation." We have demonstrated that capacitation is associated with an increase in the tyrosine phosphorylation of a subset of proteins and that these events are regulated by an HCO 3 ؊ /cAMP-dependent pathway involving protein kinase A. Capacitation is also accompanied by hyperpolarization of the sperm plasma membrane. Here we present evidence that, in addition to its role in the regulation of adenylyl cyclase, HCO 3 ؊ has a role in the regulation of plasma membrane potential in mouse sperm. Addition of HCO 3 ؊ but not Cl ؊ induces a hyperpolarizing current in mouse sperm plasma membranes. This HCO 3 ؊ -dependent hyperpolarization was not observed when Na ؉ was replaced by the nonpermeant cation choline ؉ . Replacement of Na ؉ by choline ؉ also inhibited the capacitation-associated increase in protein tyrosine phosphorylation as well as the zona pellucida-induced acrosome reaction. The lack of an increase in protein tyrosine phosphorylation was overcome by the presence of cAMP agonists in the incubation medium. The lack of a hyperpolarizing HCO 3 ؊ current and the inhibition of the capacitation-dependent increase in protein tyrosine phosphorylation in the absence of Na ؉ suggest that a Na ؉ /HCO 3 ؊ cotransporter is present in mouse sperm and is coupled to events regulating capacitation.Upon ejaculation, mammalian sperm are not able to fertilize; they become fertilization-competent during transit through the female reproductive tract (1). The molecular, biochemical, and physiological changes that occur in sperm while in the female tract are collectively referred to as capacitation. During capacitation, changes in membrane dynamics and properties, enzyme activities, and motility render spermatozoa responsive to stimuli that induce the acrosome reaction and prepare these cells for penetration of the egg vestments prior to fertilization. Mammalian sperm capacitation is also accompanied by the hyperpolarization of the sperm plasma membrane (3). Hyperpolarization is observed as an increase in the intracellular negative charges when compared with the extracellular environment. Although it is not clear how sperm plasma membrane potential is regulated during capacitation, it appears that membrane hyperpolarization may be partially because of an enhanced K ϩ permeability as a result of a decrease in inhibitory modulation of K ϩ channels (3). Recently, Muñ oz-Garay et al. (4) demonstrated with patch clamp techniques that inward rectifying K ϩ channels are expressed in mouse spermatogenic cells and proposed that these channels may be responsible for the capacitation-associated membrane hyperpolarization. Interestingly, Ba 2ϩ blocks these K ϩ channels with an IC 50 similar to that shown to inhibit ...

In a process called capacitation, mammalian sperm gain the ability to fertilize after residing in the female tract. During capacitation the mouse sperm plasma membrane potential (E m ) hyperpolarizes. However, the mechanisms that regulate sperm E m are not well understood. Here we show that sperm hyperpolarize when external Mammalian sperm are not able to fertilize after ejaculation. They acquire this ability only after residing in the female uterine tract for a finite period of time that varies depending on the species. The molecular, biochemical, and physiological changes that occur in sperm while in the female tract are collectively referred to as capacitation (1). Capacitation is associated with changes in membrane properties, enzyme activities, and motility that prepare the sperm for the acrosome reaction and for penetration of the egg vestments prior to fertilization. The molecular basis of capacitation has been partially defined and includes: the removal of cholesterol from the sperm plasma membrane by cholesterol acceptors such as bovine serum albumin (2, 3), modifications in plasma membrane phospholipids, fluxes of HCO 3 Ϫ (4) and other intracellular ions, and increased tyrosine phosphorylation of proteins (5-7). These events are likely to play a role in the induction of hyperactivated motility and the ability of the sperm to undergo a regulated acrosome reaction (for review see Ref. 8).Bovine and mouse sperm capacitation is also accompanied by a plasma membrane hyperpolarization. E m decreases in mouse sperm from Ϫ38 to Ϫ55 mV (4, 9, 10) and in bovine sperm from Ϫ33 to Ϫ66 mV (9). Because capacitation prepares sperm for the acrosome reaction, the capacitation-associated hyperpolarization may regulate the ability of sperm to generate transient Ca 2ϩ elevations during the acrosome reaction induced by physiological agonists (e.g. zona pellucida) (11). In this respect, low voltage-activated T-type Ca 2ϩ channels have been detected in mouse spermatogenic cells (12, 13), and these channels are also present in mature mouse sperm (14, 15). One unique property of low voltage-activated Ca 2ϩ channels is that they inactivate at the resting E m of sperm prior to capacitation (around Ϫ35 mV) (12,14). Thus, if low voltage-activated Ca 2ϩ channels are involved in the regulation of the acrosome reaction, the capacitation-associated sperm hyperpolarization may be required to remove this inactivation (11,16,17).Although the molecular mechanisms by which the sperm E m hyperpolarizes during capacitation are not clear, there exist several potential candidates. demonstrated with patch clamp techniques that inward rectifying K ϩ channels are expressed in mouse spermatogenic cells and proposed that these channels may contribute to the capacitation-associated sperm membrane hyperpolarization. An increase in sperm K ϩ permeability should lead to an E m hyperpolarization, according to the K ϩ equilibrium potential (18). Alternatively, the sperm plasma membrane may become less permeable to Na ϩ . The relatively depolarized mamma...