The nuclear matrix has been implicated in several cellular processes, including DNA replication, transcription, and RNA processing. In particular, transcriptional regulation is believed to be accomplished by binding of chromatin loops to the nuclear matrix and by the concentration of specific transcription factors near these matrix attachment regions (MARs). A number of MAR-binding proteins have been identified, but few have been directly linked to tissue-specific transcription. Recently, we have identified two cellular protein complexes (NBP and UBP) that bind to a region of the mouse mammary tumor virus (MMTV) long terminal repeat (LTR) previously shown to contain at least two negative regulatory elements (NREs) termed the promoter-proximal and promoter-distal NREs. These NREs are absent from MMTV strains that cause T-cell lymphomas instead of mammary carcinomas. We show here that NBP binds to a 22-bp sequence containing an imperfect inverted repeat in the promoter-proximal NRE. Previous data showed that a mutation (p924) within the inverted repeat elevated basal transcription from the MMTV promoter and destabilized the binding of NBP, but not UBP, to the proximal NRE. By using conventional and affinity methods to purify NBP from rat thymic nuclear extracts, we obtained a single major protein of 115 kDa that was identified by protease digestion and partial sequencing analysis as the nuclear matrix-binding protein special AT-rich sequence-binding protein 1 (SATB1). Antibody ablation, distamycin inhibition of binding, renaturation and competition experiments, and tissue distribution data all confirmed that the NBP complex contained SATB1. Similar types of experiments were used to show that the UBP complex contained the homeodomain protein Cux/CDP that binds the MAR of the intronic heavy-chain immunoglobulin enhancer. By using the p924 mutation within the MMTV LTR upstream of the chloramphenicol acetyltransferase gene, we generated two strains of transgenic mice that had a dramatic elevation of reporter gene expression in lymphoid tissues compared with reporter gene expression in mice expressing wild-type LTR constructs. Thus, the 924 mutation in the SATB1-binding site dramatically elevated MMTV transcription in lymphoid tissues. These results and the ability of the proximal NRE in the MMTV LTR to bind to the nuclear matrix clearly demonstrate the role of MAR-binding proteins in tissue-specific gene regulation and in MMTV-induced oncogenesis.The nuclear matrix is a network of RNA and nonhistone proteins that serves as a scaffold for loops of chromatin (10,11,64). This matrix has been associated with the regulation of DNA replication, transcription, and RNA processing (for a review, see reference 64). The DNA regions anchoring chromatin to the matrix (called matrix-associated regions [MARs] or scaffold-associated regions) (18) are composed of AT-rich sequences that have a unique structure characterized by high unwinding potential and the formation of a narrow minor groove (1, 52). MARs have been shown to colocal...

Bipolar cells are the central neurons of the retina that transmit visual signals from rod and cone photoreceptors to third-order neurons in the inner retina and the brain. A dogma set forth by early anatomical studies is that bipolar cells in mammalian retinas receive segregated rod/cone synaptic inputs (either from rods or from cones), and here, we present evidence that challenges this traditional view. By analyzing light-evoked cation currents from morphologically identified depolarizing bipolar cells (DBCs) in the wild-type and three pathway-specific knockout mice (rod transducin knockout [Trα], and transcription factor beta4 knockout [Bhlhb4, we show that a subpopulation of rod DBCs (DBC R2 s) receives substantial input directly from cones and a subpopulation of cone DBCs (DBC C1 s) receives substantial input directly from rods. These results provide evidence of the existence of functional rod-DBC C and cone-DBC R synaptic pathways in the mouse retina as well as the previously proposed rod hyperpolarizing bipolar-cells pathway. This is grounds for revising the mammalian rod/cone bipolar cell dogma.light-evoked cation and chloride currents | axon-terminal stratification | connexin36 | wild-type and mutant mice | depolarizing bipolar cell I n the visual system, rod photoreceptors register dim light signals, and cone photoreceptors encode brighter light signals (1). Bipolar cells (BCs) are the second-order neurons in the retina that receive light-elicited signals from rod and cone photoreceptors and transmit them to amacrine cells (ACs) and ganglion cells (GCs) in the inner retina (1, 2). Early anatomical studies have shown that mammalian rods make synaptic contacts with only one type of bipolar cell, the rod depolarizing bipolar cell (DBC R ), whereas cones make synaptic contacts with eight to nine types of cone depolarizing (DBC C s) or hyperpolarizing bipolar cells (HBC C s) (3-5). Additionally, DBC R s do not make output synapses directly on GCs, the output neurons of the retina, but on the AII amacrine cells (AIIACs), which make electrical synapses (with connexin36 at least at the AIIAC side) (6, 7) with DBC C s (that send signals to ON GCs) and inhibitory glycinergic synapses with HBC C s and OFF GCs (8-10). Therefore, in addition to direct cone synaptic inputs, DBC C s receive rod-mediated signals from AIIAC-DBC C electrical synapses, and HBC C s receive rod-mediated signals from the AIIAC-HBC C chemical synapses (11,12). This AIIAC-mediated rod/cone signal mixing is named the primary rod-to-cone signaling pathway (13). Furthermore, rods and cones are electrically coupled with each other, possibly through connexin36-mediated gap junctions (6,14,15), and such rod/cone-signal mixing at the photoreceptor level is named the secondary rod-cone pathway (13).The rod and cone bipolar cell-signaling circuitry described above has been considered for many years as the general organizational plan (to a certain degree as the dogma) for all mammals (3,5,16). Evidence from recent studies, however, begins to challenge t...

Retinal bipolar cells are essential to the transmission of light information. Although bipolar cell dysfunction can result in blindness, little is known about the factors required for bipolar cell development and functional maturation. The basic helix-loop-helix (bHLH) transcription factor Bhlhb4 was found to be expressed in rod bipolar cells (RB). Electroretinograms (ERGs) in the adult Bhlhb4 knockout (Bhlhb4(-/-)) showed that the loss of Bhlhb4 resulted in disrupted rod signaling and profound retinal dysfunction resembling human congenital stationary night blindness (CSNB), characterized by the loss of the scotopic ERG b-wave. A depletion of inner nuclear layer (INL) cells in the adult Bhlhb4 knockout has been ascribed to the abolishment of the RB cell population during postnatal development. Other retinal cell populations including photoreceptors were unaltered. The timing of RB cell depletion in the Bhlhb4(-/-) mouse suggests that Bhlhb4 is essential for RB cell maturation.

A monumental task of the mammalian retina is to encode an enormous range (>10(9)-fold) of light intensities experienced by the animal in natural environments. Retinal neurons carry out this task by dividing labor into many parallel rod and cone synaptic pathways. Here we study the operational plan of various rod- and cone-mediated pathways by analyzing electroretinograms (ERGs), primarily b-wave responses, in dark-adapted wildtype, connexin36 knockout, depolarizing rod-bipolar cell (DBCR) knockout, and rod transducin alpha-subunit knockout mice [WT, Cx36(-/-), Bhlhb4(-/-), and Tralpha(-/-)]. To provide additional insight into the cellular origins of various components of the ERG, we compared dark-adapted ERG responses with response dynamic ranges of individual retinal cells recorded with patch electrodes from dark-adapted mouse retinas published from other studies. Our results suggest that the connexin36-mediated rod-cone coupling is weak when light stimulation is weak and becomes stronger as light stimulation increases in strength and that rod signals may be transmitted to some DBCCs via direct chemical synapses. Moreover, our analysis indicates that DBCR responses contribute about 80% of the overall DBC response to scotopic light and that rod and cone signals contribute almost equally to the overall DBC responses when stimuli are strong enough to saturate the rod bipolar cell response. Furthermore, our study demonstrates that analysis of ERG b-wave of dark-adapted, pathway-specific mutants can be used as an in vivo tool for dissecting rod and cone synaptic pathways and for studying the functions of pathway-specific gene products in the retina.